Secreted and transmembrane polypeptides and nucleic acids encoding the same

ABSTRACT

The present invention is directed to novel polypeptides and to nucleic acid molecules encoding those polypeptides. Also provided herein are vectors and host cells comprising those nucleic acid sequences, chimeric polypeptide molecules comprising the polypeptides of the present invention fused to heterologous polypeptide sequences, antibodies which bind to the polypeptides of the present invention and to methods for producing the polypeptides of the present invention.

CROSS REFERENCE TO RELATED APPLICATIONS

[0001] This is a continuation application claiming priority under 35 USC§120 to U.S. Ser. No. 10/006867 Filed Dec. 6, 2001, and which claimspriority under 35 USC §119 to U.S. provisional serial No. 60/063,435Filed Oct. 29, 1997; No. 60/064,215 Filed Oct. 29, 1997; No. 60/082,797Filed Apr. 22, 1998; No. 60/083,495 Filed Apr. 29, 1998; No. 60/085,579Filed May 15, 1998; No. 60/087,759 Filed Jun. 2, 1998; No. 60/088,021Filed Jun. 4, 1998; No. 60/088,029 Filed Jun. 4, 1998; No. 60/088,030Filed Jun. 4, 1998; No. 60/088,734 Filed Jun. 10, 1998; No. 60/088,740Filed Jun. 10, 1998; No. 60/088,811 Filed Jun. 10, 1998; No. 60/088,824Filed Jun. 10, 1998; No. 60/088,825 Filed Jun. 10, 1998; No. 60/088,863Filed Jun. 11, 1998; No. 60/089,105 Filed Jun. 12, 1998; No. 60/089,514Filed Jun. 16, 1998; No. 60/089,653 Filed Jun. 17, 1998; No. 60/089,952Filed Jun. 19, 1998; No. 60/090,246 Filed Jun. 22, 1998; No. 60/090,444Filed Jun. 24, 1998; No. 60/090,688 Filed Jun. 25, 1998; No. 60/090,696Filed Jun. 25, 1998; No. 60/090,862 Filed Jun. 26, 1998; No. 60/091,628Filed Jul. 2, 1998; No. 60/096,012 Filed Aug. 10, 1998; No. 60/096,757Filed Aug. 17, 1998; No. 60/096,949 Filed Aug. 18, 1998; No. 60/096,959Filed Aug. 18, 1998; No. 60/097,954 Filed Aug. 26, 1998; No. 60/097,971Filed Aug. 26, 1998; No. 60/097,979 Filed Aug. 26, 1998; No. 60/098,749Filed Sep. 1, 1998; No. 60/099,741 Filed Sep. 10, 1998; No. 60/099,763Filed Sep. 10, 1998; No. 60/099,792 Filed Sep. 10, 1998; No. 60/099,812Filed Sep. 10, 1998; No. 60/099,815 Filed Sep. 10, 1998; No. 60/100,627Filed Sep. 16, 1998; No. 60/100,662 Filed Sep. 16, 1998; No. 60/100,683Filed Sep. 17, 1998; No. 60/100,684 Filed Sep. 17, 1998; No. 60/100,930Filed Sep. 17, 1998; No. 60/101,279 Filed Sep. 22, 1998; No. 60/101,475Filed Sep. 23, 1998; No. 60/101,738 Filed Sep. 24, 1998; No. 60/101,743Filed Sep. 24, 1998; No. 60/101,916 Filed Sep. 24, 1998; No. 60/102,570Filed Sep. 30, 1998; No. 60/103,449 Filed Oct. 6, 1998; No. 60/103,678Filed Oct. 8, 1998; No. 60/103,679 Filed Oct. 8, 1998; No. 60/103,711Filed Oct. 8, 1998; No. 60/105,000 Filed Oct. 20, 1998; No. 60/105,002Filed Oct. 20, 1998; No. 60/105,881 Filed Oct. 27, 1998; No. 60/106,030Filed Oct. 28, 1998; No. 60/106,464 Filed Oct. 30, 1998; No. 60/106,856Filed Nov. 3, 1998; No. 60/108,807 Filed Nov. 17, 1998; No. 60/112,419Filed Dec. 15, 1998; No. 60/112,422 Filed Dec. 15, 1998; No. 60/112,853Filed Dec. 16, 1998; No. 60/113,011 Filed Dec. 16, 1998; No. 60/112,854Filed Dec. 16, 1998; No. 60/113,300 Filed Dec. 22, 1998; No. 60/113,408Filed Dec. 22, 1998; No. 60/113,430 Filed Dec. 23, 1998; No. 60/113,621Filed Dec. 23, 1998; No. 60/114,223 Filed Dec. 30, 1998; No. 60/115,614Filed Jan. 12, 1999; No. 60/116,527 Filed Jan. 20, 1999; No. 60/116,843Filed Jan. 22, 1999; No. 60/119,285 Filed Feb. 9, 1999; No. 60/119,287Filed Feb. 9, 1999; No. 60/119,525 Filed Feb. 10, 1999; No. 60/119,549Filed Feb. 10, 1999; No. 60/120,014 Filed Feb. 11, 1999; No. 60/129,122Filed Apr. 13, 1999; No. 60/129,674 Filed Apr. 16, 1999; No. 60/131,291Filed Apr. 27, 1999; No. 60/138,387 Filed Jun. 9, 1999; No. 60/1144,791Filed Jul. 20, 1999; No. 60/169,495 Filed Dec. 7, 1999; No. 60/175,481Filed Jan. 11, 2000; No. 60,/191,007 Filed Mar. 21, 2000; No. 60/199,397Filed Apr. 25, 2000 and which claims priority under 35 USC §120 to USPatent Applications and PCT International patent application Ser. No.09/380,139 Filed Aug. 25, 1999; Ser. No. 09/311,832 Filed May 14, 1999;Ser. No. 09/380,137 Filed Aug. 25, 1999, now abandoned; 09/380,138 FiledAug. 25, 1999, now abandoned; 09/380,142 Filed Aug. 25, 1999, nowabandoned; 09/397,342 Filed Sep. 15, 1999; Ser. No. 09/403,297 FiledOct. 18, 1999, now abandoned; 09/423,844 Filed Nov. 12, 1999, nowabandoned; 109/644,848 Filed Aug. 22, 2000; Ser. No. 09/665,350 FiledSep. 18, 2000; Ser. No. 09/664,610 Filed Sep. 18, 2000, now abandoned;09/709,238 Filed Nov. 8, 2000; Ser. No. 09/747,259 Filed Dec. 20, 2000;Ser. No. 09/816,744 Filed Mar. 22, 2001; Ser. No. 09/854,208 Filed May10, 2001; Ser. No. 09/854,280 Filed May 10, 2001; Ser. No. 09/870,574Filed May 30, 2001; Ser. No. 09/874,503 Filed Jun. 5, 2001; Ser. No.09/908,827 Filed Jul. 18, 2001; Ser. No. 09/869,566 Filed Feb. 19, 2002;PCT/US98/19330 Filed Sep. 16, 1998; PCT/US99/05028 Filed Mar. 8, 1999;PCT/US99/10733 Filed May 14, 1999; PCT/US99/12252 Filed Jun. 2, 1999;PCT/US99/20111 Filed Sep. 1, 1999; PCT/US99/21090 Filed Sep. 15, 1999;PCT/US99/21194 Filed Sep. 15, 1999; PCT/US99/30720 Filed Dec. 22, 1999;PCT/US00/04341 Filed Feb. 18, 2000; PCT/US00/04342 Filed Feb. 18, 2000;PCT/US00/04414 Filed Feb. 22, 2000; PCT/US00/05601 Filed 3/1/O;PCT/US00/08439 Filed Mar. 30, 2000; PCT/US00/14042 Filed May 22, 2000;PCT/US00/15264 Filed Jun. 2, 2000; PCT/US00/23522 Filed Aug. 23, 2000;PCT/US00/23328 Filed Aug. 24, 2000; PCT/US00/30873 Filed Jan. 10, 2000;PCT/US00/32678 Filed Dec. 1, 2000; PCT/US00/34956 Filed Dec. 20, 2000;PCT/US01/06520 Filed Feb. 28, 2001; PCT/US01/06666 Filed Mar. 1, 2001;PCT/US01/17443 Filed May 30, 2001; PCT/US01/17800 Filed Jun. 1, 2001;PCT/US01/19692 Filed Jun. 20, 2001; PCT/US01/21066 Filed Jun. 29, 2001;PCT/US01/21735 Filed Jul. 9, 2001, the entire disclosures of which arehereby incorporated by reference.

BACKGROUND OF INVENTION

[0002] The present invention relates generally to the identification andisolation of novel DNA and to the recombinant production of novelpolypeptides.

[0003] Extracellular proteins play important roles in, among otherthings, the formation, differentiation and maintenance of multicellularorganisms. The fate of many individual cells, e.g., proliferation,migration, differentiation, or interaction with other cells, istypically governed by information received from other cells and/or theimmediate environment. This information is often transmitted by secretedpolypeptides (for instance, mitogenic factors, survival factors,cytotoxic factors, differentiation factors, neuropeptides, and hormones)which are, in turn, received and interpreted by diverse cell receptorsor membrane-bound proteins. These secreted polypeptides or signalingmolecules normally pass through the cellular secretory pathway to reachtheir site of action in the extracellular environment.

[0004] Secreted proteins have various industrial applications, includingas pharmaceuticals, diagnostics, biosensors and bioreactors. Mostprotein drugs available at present, such as thrombolytic agents,interferons, interleukins, erythropoietins, colony stimulating factors,and various other cytokines, are secretory proteins. Their receptors,which are membrane proteins, also have potential as therapeutic ordiagnostic agents. Efforts are being undertaken by both industry andacademia to identify new, native secreted proteins. Many efforts arefocused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel secreted proteins. Examples ofscreening methods and techniques are described in the literature [see,for example, Klein et al., Proc. Natl. Acad. Sci. 93:7108-7113 (1996);U.S. Pat. No. 5,536,637)].

[0005] Membrane-bound proteins and receptors can play important rolesin, among other things, the formation, differentiation and maintenanceof multicellular organisms. The fate of many individual cells, e.g.,proliferation, migration, differentiation, or interaction with othercells, is typically governed by information received from other cellsand/or the immediate environment. This information is often transmittedby secreted polypeptides (for instance, mitogenic factors, survivalfactors, cytotoxic factors, differentiation factors, neuropeptides, andhormones) which are, in turn, received and interpreted by diverse cellreceptors or membrane-bound proteins. Such membrane-bound proteins andcell receptors include, but are not limited to, cytokine receptors,receptor kinases, receptor phosphatases, receptors involved in cell-cellinteractions, and cellular adhesin molecules like selectins andintegrins. For instance, transduction of signals that regulate cellgrowth and differentiation is regulated in part by phosphorylation ofvarious cellular proteins. Protein tyrosine kinases, enzymes thatcatalyze that process, can also act as growth factor receptors. Examplesinclude fibroblast growth factor receptor and nerve growth factorreceptor.

[0006] Membrane-bound proteins and receptor molecules have variousindustrial applications, including as pharmaceutical and diagnosticagents. Receptor immunoadhesins, for instance, can be employed astherapeutic agents to block receptor-ligand interactions. Themembrane-bound proteins can also be employed for screening of potentialpeptide or small molecule inhibitors of the relevant receptor/ligandinteraction.

[0007] Efforts are being undertaken by both industry and academia toidentify new, native receptor or membrane-bound proteins. Many effortsare focused on the screening of mammalian recombinant DNA libraries toidentify the coding sequences for novel receptor or membrane-boundproteins.

SUMMARY OF INVENTION

[0008] In one embodiment, the invention provides an isolated nucleicacid molecule comprising a nucleotide sequence that encodes a PROpolypeptide.

[0009] In one aspect, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule encoding a PRO polypeptide having afull-length amino acid sequence as disclosed herein, an amino acidsequence lacking the signal peptide as disclosed herein, anextracellular domain of a transmembrane protein, with or without thesignal peptide, as disclosed herein or any other specifically definedfragment of the full-length amino acid sequence as disclosed herein, or(b) the complement of the DNA molecule of (a).

[0010] In other aspects, the isolated nucleic acid molecule comprises anucleotide sequence having at least about 80% nucleic acid sequenceidentity, alternatively at least about 81% nucleic acid sequenceidentity, alternatively at least about 82% nucleic acid sequenceidentity, alternatively at least about 83% nucleic acid sequenceidentity, alternatively at least about 84% nucleic acid sequenceidentity, alternatively at least about 85% nucleic acid sequenceidentity, alternatively at least about 86% nucleic acid sequenceidentity, alternatively at least about 87% nucleic acid sequenceidentity, alternatively at least about 88% nucleic acid sequenceidentity, alternatively at least about 89% nucleic acid sequenceidentity, alternatively at least about 90% nucleic acid sequenceidentity, alternatively at least about 91% nucleic acid sequenceidentity, alternatively at least about 92% nucleic acid sequenceidentity, alternatively at least about 93% nucleic acid sequenceidentity, alternatively at least about 94% nucleic acid sequenceidentity, alternatively at least about 95% nucleic acid sequenceidentity, alternatively at least about 96% nucleic acid sequenceidentity, alternatively at least about 97% nucleic acid sequenceidentity, alternatively at least about 98% nucleic acid sequenceidentity and alternatively at least about 99% nucleic acid sequenceidentity to (a) a DNA molecule comprising the coding sequence of afull-length PRO polypeptide cDNA as disclosed herein, the codingsequence of a PRO polypeptide lacking the signal peptide as disclosedherein, the coding sequence of an extracellular domain of atransmembrane PRO polypeptide, with or without the signal peptide, asdisclosed herein or the coding sequence of any other specificallydefined fragment of the full-length amino acid sequence as disclosedherein, or (b) the complement of the DNA molecule of (a).

[0011] In a further aspect, the invention concerns an isolated nucleicacid molecule comprising a nucleotide sequence having at least about 80%nucleic acid sequence identity, alternatively at least about 81% nucleicacid sequence identity, alternatively at least about 82% nucleic acidsequence identity, alternatively at least about 83% nucleic acidsequence identity, alternatively at least about 84% nucleic acidsequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively it least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98% nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity to (a) a DNA molecule that encodes the same maturepolypeptide encoded by any of the human protein cDNAs deposited with theATCC as disclosed herein, or (b) the complement of the DNA molecule of(a).

[0012] Another aspect the invention provides an isolated nucleic acidmolecule comprising a nucleotide sequence encoding a PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated, or is complementary to such encoding nucleotidesequence, wherein the transmembrane domain(s) of such polypeptide aredisclosed herein. Therefore, soluble extracellular domains of the hereindescribed PRO polypeptides are contemplated.

[0013] Another embodiment is directed to fragments of a PRO polypeptidecoding sequence, or the complement thereof, that may find use as, forexample, hybridization probes, for encoding fragments of a PROpolypeptide that may optionally encode a polypeptide comprising abinding site for an anti-PRO antibody or as antisense oligonucleotideprobes. Such nucleic acid fragments are usually at least about 20nucleotides in length, alternatively at least about 30 nucleotides inlength, alternatively at least about 40 nucleotides in length,alternatively at least about 50 nucleotides in length, alternatively atleast about 60 nucleotides in length, alternatively at least about 70nucleotides in length, alternatively at least about 80 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 100 nucleotides in length, alternatively atleast about 110 nucleotides in length, alternatively at least about 120nucleotides in length, alternatively at least about 130 nucleotides inlength, alternatively at least about 140 nucleotides in length,alternatively at least about 150 nucleotides in length, alternatively atleast about 160 nucleotides in length, alternatively at least about 170nucleotides in length, alternatively at least about 180 nucleotides inlength, alternatively sat least about 190 nucleotides in length,alternatively at least about 200 nucleotides in length, alternatively atleast about 250 nucleotides in length, alternatively at least about 300nucleotides in length, alternatively at least about 350 nucleotides inlength, alternatively at least about 400 nucleotides in length,alternatively at least about 450 nucleotides in length, alternatively atleast about 500 nucleotides in length, alternatively at least about 600nucleotides in length, alternatively at least about 700 nucleotides inlength, alternatively at least about 800 nucleotides in length,alternatively at least about 900 nucleotides in length and alternativelyat least about 1000 nucleotides in length, wherein in this context theterm “about” means the referenced nucleotide sequence length plus orminus 10% of that referenced length. It is noted that novel fragments ofa PRO polypeptide-encoding nucleotide sequence may be determined in aroutine manner by aligning the PRO polypeptide-encoding nucleotidesequence with other known nucleotide sequences using any of a number ofwell known sequence alignment programs and determining which PROpolypeptide-encoding nucleotide sequence fragment(s) are novel. All ofsuch PRO polypeptide-encoding nucleotide sequences are contemplatedherein. Also contemplated are the PRO polypeptide fragments encoded bythese nucleotide molecule fragments, preferably those PRO polypeptidefragments that comprise a binding site for an anti-PRO antibody.

[0014] In another embodiment, the invention provides isolated PROpolypeptide encoded by any of the isolated nucleic acid sequenceshereinabove identified.

[0015] In a certain aspect, the invention concerns an isolated PROpolypeptide, comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to a PROpolypeptide having a full-length amino acid sequence as disclosedherein, an amino acid sequence lacking the signal peptide as disclosedherein, an extracellular domain of a transmembrane protein, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of the full-length amino acid sequence asdisclosed herein.

[0016] In a further aspect, the invention concerns an isolated PROpolypeptide comprising an amino acid sequence having at least about 80%amino acid sequence identity, alternatively at least about 81% aminoacid sequence identity, alternatively at least about 82% amino acidsequence identity, alternatively at least about 83% amino acid sequenceidentity, alternatively at least about 84% amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88% amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92% amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 96% amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to anamino acid sequence encoded by any of the human protein cDNAs depositedwith the ATCC as disclosed herein.

[0017] In a specific aspect, the invention provides an isolated PROpolypeptide without the N-terminal signal sequence and/or the initiatingmethionine and is encoded by a nucleotide sequence that encodes such anamino acid sequence as hereinbefore described. Processes for producingthe same are also herein described, wherein those processes compriseculturing a host cell comprising a vector which comprises theappropriate encoding nucleic acid molecule under conditions suitable forexpression of the PRO polypeptide and recovering the PRO polypeptidefrom the cell culture.

[0018] Another aspect the invention provides an isolated PRO polypeptidewhich is either transmembrane domain-deleted or transmembranedomain-inactivated. Processes for producing the same are also hereindescribed, wherein those processes comprise culturing a host cellcomprising a vector which comprises the appropriate encoding nucleicacid molecule under conditions suitable for expression of the PROpolypeptide and recovering the PRO polypeptide from the cell culture.

[0019] In yet another embodiment, the invention concerns agonists andantagonists of a native PRO polypeptide as defined herein. In aparticular embodiment, the agonist or antagonist is an anti-PRO antibodyor a small molecule.

[0020] In a further embodiment, the invention concerns a method ofidentifying agonists or antagonists to a PRO polypeptide which comprisecontacting the PRO polypeptide with a candidate molecule and monitoringa biological activity mediated by said PRO polypeptide. Preferably, thePRO polypeptide is a native PRO polypeptide.

[0021] In a still further embodiment, the invention concerns acomposition of matter comprising a PRO polypeptide, or an agonist orantagonist of a PRO polypeptide as herein described, or an anti-PROantibody, in combination with a carrier. Optionally, the carrier is apharmaceutically acceptable carrier.

[0022] Another embodiment of the present invention is directed to theuse of a PRO polypeptide, or an agonist or antagonist thereof ashereinbefore described, or an anti-PRO antibody, for the preparation ofa medicament useful in the treatment of a condition which is responsiveto the PRO polypeptide, an agonist or antagonist thereof or an anti-PROantibody.

[0023] In other embodiments of the present invention, the inventionprovides vectors comprising DNA encoding any of the herein describedpolypeptides. Host cell comprising any such vector are also provided. Byway of example, the host cells may be CHO cells, E. coli, or yeast. Aprocess for producing any of the herein described polypeptides isfurther provided and comprises culturing host cells under conditionssuitable for expression of the desired polypeptide and recovering thedesired polypeptide from the cell culture.

[0024] In other embodiments, the invention provides chimeric moleculescomprising any of the herein described polypeptides fused to aheterologous polypeptide or amino acid sequence. Example of suchchimeric molecules comprise any of the herein described polypeptidesfused to an epitope tag sequence or a Fc region of an immunoglobulin.

[0025] In another embodiment, the invention provides an antibody whichbinds, preferably specifically, to any of the above or below describedpolypeptides. Optionally, the antibody is a monoclonal antibody,humanized antibody, antibody fragment or single-chain antibody.

[0026] In yet other embodiments, the invention provides oligonucleotideprobes useful for isolating genomic and cDNA nucleotide sequences or asantisense probes, wherein those probes may be derived from any of theabove or below described nucleotide sequences.

[0027] In yet other embodiments, the present invention is directed tomethods of using the PRO polypeptides of the present invention for avariety of uses based upon the functional biological assay datapresented in the Examples below.

BRIEF DESCRIPTION OF DRAWINGS

[0028]FIG. 1 shows a nucleotide sequence (SEQ ID NO:1) of a nativesequence PRO180 cDNA, wherein SEQ ID NO:1 is a clone designated hereinas “DNA26843-1389”.

[0029]FIG. 2 shows the amino acid sequence (SEQ ID NO:2) derived fromthe coding sequence of SEQ ID NO:1 shown in FIG. 1.

[0030]FIG. 3 shows a nucleotide sequence (SEQ ID NO:3) of a nativesequence PRO218 cDNA, wherein SEQ ID NO:3 is a clone designated hereinas “DNA30867-1335”.

[0031]FIG. 4 shows the amino acid sequence (SEQ ID NO:4) derived fromthe coding sequence of SEQ ID NO:3 shown in FIG. 3.

[0032]FIG. 5 shows a nucleotide sequence (SEQ ID NO:5) of a nativesequence PRO263 cDNA, wherein SEQ ID NO:5 is a clone designated hereinas “DNA34431-1177”.

[0033]FIG. 6 shows the amino acid sequence (SEQ ID NO:6) derived fromthe coding sequence of SEQ ID NO:5 shown in FIG. 5.

[0034]FIG. 7 shows a nucleotide sequence (SEQ ID NO:7) of a nativesequence PRO295 cDNA, wherein SEQ ID NO:7 is a clone designated hereinas “DNA38268-1188”.

[0035]FIG. 8 shows the amino acid sequence (SEQ ID NO:8) derived fromthe coding sequence of SEQ ID NO:7 shown in FIG. 7.

[0036]FIG. 9 shows a nucleotide sequence (SEQ ID NO:9) of a nativesequence PRO874 cDNA, wherein SEQ ID NO:9 is a clone designated hereinas “DNA40621-1440”.

[0037]FIG. 10 shows the amino acid sequence (SEQ ID NO:10) derived fromthe coding sequence of SEQ ID NO:9 shown in FIG. 9.

[0038]FIG. 11 shows a nucleotide sequence (SEQ ID NO:11) of a nativesequence PRO300 cDNA, wherein SEQ ID NO:11 is a clone designated hereinas “DNA40625-1189”.

[0039]FIG. 12 shows the amino acid sequence (SEQ ID NO:12) derived fromthe coding sequence of SEQ ID NO:11 shown in FIG. 11.

[0040]FIG. 13 shows a nucleotide sequence (SEQ ID NO: 13) of a nativesequence PRO1864 cDNA, wherein SEQ ID NO:13 is a clone designated hereinas “DNA45409-2511”.

[0041]FIG. 14 shows the amino acid sequence (SEQ ID NO:14) derived fromthe coding sequence of SEQ ID NO:13 shown in FIG. 13.

[0042]FIG. 15 shows a nucleotide sequence (SEQ ID NO: 15) of a nativesequence PRO1282 cDNA, wherein SEQ ID NO:15 is a clone designated hereinas “DNA45495-1550”.

[0043]FIG. 16 shows the amino acid sequence (SEQ ID NO:16) derived fromthe coding sequence of SEQ ID NO:15 shown in FIG. 15.

[0044]FIG. 17 shows a nucleotide sequence (SEQ ID NO:17) of a nativesequence PRO1063 cDNA, wherein SEQ ID NO:17 is a clone designated hereinas “DNA49820-1427”.

[0045]FIG. 18 shows the amino acid sequence (SEQ ID NO:18) derived fromthe coding sequence of SEQ ID NO:17 shown in FIG. 17.

[0046]FIG. 19 shows a nucleotide sequence (SEQ ID NO: 19) of a nativesequence PRO1773 cDNA, wherein SEQ ID NO:19 is a clone designated hereinas “DNA56406-1704”.

[0047]FIG. 20 shows the amino acid sequence (SEQ ID NO:20) derived fromthe coding sequence of SEQ ID NO:19 shown in FIG. 19.

[0048]FIG. 21 shows a nucleotide sequence (SEQ ID NO:21) of a nativesequence PRO01013 cDNA, wherein SEQ ID NO:21 is a clone designatedherein as “DNA56410-1414”.

[0049]FIG. 22 shows the amino acid sequence (SEQ ID NO:22) derived fromthe coding sequence of SEQ ID NO:21 shown in FIG. 21.

[0050]FIG. 23 shows a nucleotide sequence (SEQ ID NO:23) of a nativesequence PRO937 cDNA, wherein SEQ ID NO:23 is a clone designated hereinas “DNA56436-1448”.

[0051]FIG. 24 shows the amino acid sequence (SEQ ID NO:24) derived fromthe coding sequence of SEQ ID NO:23 shown in FIG. 23.

[0052]FIG. 25 shows a nucleotide sequence (SEQ ID NO:25) of a nativesequence PRO842 cDNA, wherein SEQ ID NO:25 is a clone designated hereinas “DNA568SS-1447”.

[0053]FIG. 26 shows the amino acid sequence (SEQ ID NO:26) derived fromthe coding sequence of SEQ ID NO:25 shown in FIG. 25.

[0054]FIG. 27 shows a nucleotide sequence (SEQ ID NO:27) of a nativesequence PRO1180 cDNA, wherein SEQ ID NO:27 is a clone designated hereinas “DNA56860-1510”.

[0055]FIG. 28 shows the amino acid sequence (SEQ ID NO:28) derived fromthe coding sequence of SEQ ID NO:27 shown in FIG. 27.

[0056]FIG. 23 shows a nucleotide sequence (SEQ ID NO:29) of a nativesequence PRO831 cDNA, wherein SEQ ID NO:29 is a clone designated hereinas “DNA56862-1343”.

[0057]FIG. 30 shows the amino acid sequence (SEQ ID NO:30) derived fromthe coding sequence of SEQ ID NO:29 shown in FIG. 29.

[0058]FIG. 31 shows a nucleotide sequence (SEQ ID NO:31) of a nativesequence PRO1115 cDNA, wherein SEQ ID NO:31 is a clone designated hereinas “DNA56868-1478”.

[0059]FIG. 32 shows the amino acid sequence (SEQ ID NO:32) derived fromthe coding sequence of SEQ ID NO:31 shown in FIG. 31.

[0060]FIG. 33 shows a nucleotide sequence (SEQ ID NO:33) of a nativesequence PRO1277 cDNA, wherein SEQ ID NO:33 is a clone designated hereinas “DNA56869-1545”.

[0061]FIG. 34 shows the amino acid sequence (SEQ ID NO:34) derived fromthe coding sequence of SEQ ID NO:33 shown in FIG. 33.

[0062]FIG. 35 shows a nucleotide sequence (SEQ ID NO:35) of a nativesequence PRO1074 cDNA, wherein SEQ ID NO:35 is a clone designated hereinas “DNA57704-1452”.

[0063]FIG. 36 shows the amino acid sequence (SEQ ID NO:36) derived fromthe coding sequence of SEQ ID NO:35 shown in FIG. 35.

[0064]FIG. 37 shows a nucleotide sequence (SEQ ID NO:37) of a nativesequence PRO1344 cDNA, wherein SEQ ID NO:37 is a clone designated hereinas “DNA58723-1588”.

[0065]FIG. 38 shows the amino acid sequence (SEQ ID NO:38) derived fromthe coding sequence of SEQ ID NO:37 shown in FIG. 37.

[0066]FIG. 39 shows a nucleotide sequence (SEQ ID NO:39) of a nativesequence PRO1136 cDNA, wherein SEQ ID NO:39 is a clone designated hereinas “DNA57827-1493”.

[0067]FIG. 40 shows the amino acid sequence (SEQ ID NO:40) derived fromthe coding sequence of SEQ ID NO:39 shown in FIG. 39.

[0068]FIG. 41 shows a nucleotide sequence (SEQ ID NO:41) of a nativesequence PRO1109 cDNA, wherein SEQ ID NO:41 is a clone designated hereinas “DNA58737-1473”.

[0069]FIG. 42 shows the amino acid sequence (SEQ ID NO:42) derived fromthe coding sequence of SEQ ID NO:41 shown in FIG. 41.

[0070]FIG. 43 shows a nucleotide sequence (SEQ ID NO:43) of a nativesequence PRO1003 cDNA, wherein SEQ ID NO:43 is a clone designated hereinas “DNA58846-1409”.

[0071]FIG. 44 shows the amino acid sequence (SEQ ID NO:44) derived fromthe coding sequence of SEQ ID NO:43 shown in FIG. 43.

[0072]FIG. 45 shows a nucleotide sequence (SEQ ID NO:45) of a nativesequence PRO138 cDNA, wherein SEQ ID NO:45 is a clone designated hereinas “DNA58850-1495”.

[0073]FIG. 46 shows the amino acid sequence (SEQ ID NO:46) derived fromthe coding sequence of SEQ ID NO:45 shown in FIG. 45.

[0074]FIG. 47 shows a nucleotide sequence (SEQ ID NO:47) of a nativesequence PRO994 cDNA, wherein SEQ ID NO:47 is a clone designated hereinas “DNA58855-1422”.

[0075]FIG. 48 shows the amino acid sequence (SEQ ID NO:48) derived fromthe coding sequence of SEQ ID NO:47 shown in FIG. 47.

[0076]FIG. 49 shows a nucleotide sequence (SEQ ID NO:49) of a nativesequence PRO1069 cDNA, wherein SEQ ID NO:49 is a clone designated hereinas “DNA59211-1450”.

[0077]FIG. 50 shows the amino acid sequence (SEQ ID NO:50) derived fromthe coding sequence of SEQ ID NO:49 shown in FIG. 49.

[0078]FIG. 51 shows a nucleotide sequence (SEQ ID NO:51) of a nativesequence PRO411 cDNA, wherein SEQ ID NO:51 is a clone designated hereinas “DNA59212-1627”.

[0079]FIG. 52 shows the amino acid sequence (SEQ ID NO:52) derived fromthe coding sequence of SEQ ID NO:51 shown in FIG. 51.

[0080]FIG. 53 shows a nucleotide sequence (SEQ ID NO:53) of a nativesequence PRO1129 cDNA, wherein SEQ ID NO:53 is a clone designated hereinas “DNA59213-1487”.

[0081]FIG. 54 shows the amino acid sequence (SEQ ID NO:54) derived fromthe coding sequence of SEQ ID NO:53 shown in FIG. 53.

[0082]FIG. 55 shows a nucleotide sequence (SEQ ID NO:55) of a nativesequence PRO1027 cDNA, wherein SEQ ID NO:55 is a clone designated hereinas “DNA59605-1418”.

[0083]FIG. 56 shows the amino acid sequence (SEQ ID NO:56) derived fromthe coding sequence of SEQ ID NO:55 shown in FIG. 55.

[0084]FIG. 57 shows a nucleotide sequence (SEQ ID NO:57) of a nativesequence PRO1106 cDNA, wherein SEQ ID NO:57 is a clone designated hereinas “DNA59609-1470”.

[0085]FIG. 58 shows the amino acid sequence (SEQ ID NO:58) derived fromthe coding sequence of SEQ ID NO:57 shown in FIG. 57.

[0086]FIG. 59 shows a nucleotide sequence (SEQ ID NO:59) of a nativesequence PRO1291 cDNA, wherein SEQ ID NO:59 is a clone designated hereinas “DNA59610-1556”.

[0087]FIG. 60 shows the amino acid sequence (SEQ ID NO:60) derived fromthe coding sequence of SEQ ID NO:59 shown in FIG. 59.

[0088]FIG. 61 shows a nucleotide sequence (SEQ ID NO:61) of a nativesequence PRO3573 cDNA, wherein SEQ ID NO:61 is a clone designated hereinas “DNA59837-2545”.

[0089]FIG. 62 shows the amino acid sequence (SEQ ID NO:62) derived fromthe coding sequence of SEQ ID NO:61 shown in FIG. 61.

[0090]FIG. 63 shows a nucleotide sequence (SEQ ID NO:63) of a nativesequence PRO3566 cDNA, wherein SEQ ID NO:63 is a clone designated hereinas “DNA59844-2542”.

[0091]FIG. 64 shows the amino acid sequence (SEQ ID NO:64) derived fromthe coding sequence of SEQ ID NO:63 shown in FIG. 63.

[0092]FIG. 65 shows a nucleotide sequence (SEQ ID NO:65) of a nativesequence PRO1098 cDNA, wherein SEQ ID NO:65 is a clone designated hereinas “DNA59854-1459”.

[0093]FIG. 66 shows the amino acid sequence (SEQ ID NO:66) derived fromthe coding sequence of SEQ ID NO:65 shown in FIG. 65.

[0094]FIG. 67 shows a nucleotide sequence (SEQ ID NO:67) of a nativesequence PRO1158 cDNA, wherein SEQ ID NO:67 is a clone designated hereinas “DNA60625-1507”.

[0095]FIG. 68 shows the amino acid sequence (SEQ ID NO:68) derived fromthe coding sequence of SEQ ID NO:67 shown in FIG. 67.

[0096]FIG. 69 shows a nucleotide sequence (SEQ ID NO:69) of a nativesequence PRO124 cDNA, wherein SEQ ID NO:69 is a clone designated hereinas “DNA60629-1481”.

[0097]FIG. 70 shows the amino acid sequence (SEQ ID NO:70) derived fromthe coding sequence of SEQ ID NO:69 shown in FIG. 69.

[0098]FIG. 71 shows a nucleotide sequence (SEQ ID NO:71) of a nativesequence PRO1287 cDNA, wherein SEQ ID NO:71 is a clone designated hereinas “DNA61755-1554”.

[0099]FIG. 72 shows the amino acid sequence (SEQ ID NO:72) derived fromthe coding sequence of SEQ ID NO:71 shown in FIG. 71.

[0100]FIG. 73 shows a nucleotide sequence (SEQ ID NO:73) of a nativesequence PRO1335 cDNA, wherein SEQ ID NO:73 is a clone designated hereinas “DNA62812-1594”.

[0101]FIG. 74 shows the amino acid sequence (SEQ ID NO:74) derived fromthe coding sequence of SEQ ID NO:73 shown in FIG. 73.

[0102]FIG. 75 shows a nucleotide sequence (SEQ ID NO:75) of a nativesequence PRO1315 cDNA, wherein SEQ ID NO:75 is a clone designated hereinas “DNA62815-1576”.

[0103]FIG. 76 shows the amino acid sequence (SEQ ID NO:76) derived fromthe coding sequence of SEQ ID NO:75 shown in FIG. 75.

[0104]FIG. 77 shows a nucleotide sequence (SEQ ID NO: 77) of a nativesequence PRO1357 cDNA, wherein SEQ ID NO:77 is a clone designated hereinas “DNA64881-1602”.

[0105]FIG. 78 shows the amino acid sequence (SEQ ID NO:78) derived fromthe coding sequence of SEQ ID NO:77 shown in FIG. 77.

[0106]FIG. 79 shows a nucleotide sequence (SEQ ID NO:79) of a nativesequence PRO1356 cDNA, wherein SEQ ID NO:79 is a clone designated hereinas “DNA64886-1601”.

[0107]FIG. 80 shows the amino acid sequence (SEQ ID NO:80) derived fromthe coding sequence of SEQ ID NO:79 shown in FIG. 79.

[0108]FIG. 81 shows a nucleotide sequence (SEQ ID NO:81) of a nativesequence PRO1557 cDNA, wherein SEQ ID NO:81 is a clone designated hereinas “DNA64902-1667”.

[0109]FIG. 82 shows the amino acid sequence (SEQ ID NO:82) derived fromthe coding sequence of SEQ ID NO:81 shown in FIG. 81.

[0110]FIG. 83 shows a nucleotide sequence (SEQ ID NO:83) of a nativesequence PRO1347 cDNA, wherein SEQ ID NO:83 is a clone designated hereinas “DNA64950-1590”.

[0111]FIG. 84 shows the amino acid sequence (SEQ ID NO:84) derived fromthe coding sequence of SEQ ID NO:83 shown in FIG. 83.

[0112]FIG. 85 shows a nucleotide sequence (SEQ ID NO:85) of a nativesequence PRO1302 cDNA, wherein SEQ ID NO:85 is a clone designated hereinas “DNA65403-1565”.

[0113]FIG. 86 shows the amino acid sequence (SEQ ID NO:86) derived fromthe coding sequence of SEQ ID NO:85 shown in FIG. 85.

[0114]FIG. 87 shows a nucleotide sequence (SEQ ID NO:87) of a nativesequence PRO1270 cDNA, wherein SEQ ID NO:87 is a clone designated hereinas “DNA66308-1537”.

[0115]FIG. 88 shows the amino acid sequence (SEQ ID NO:88) derived fromthe coding sequence of SEQ ID NO:87 shown in FIG. 87.

[0116]FIG. 89 shows a nucleotide sequence (SEQ ID NO:89) of a nativesequence PRO1268 cDNA, wherein SEQ ID NO:89 is a clone designated hereinas “DNA66519-1535”.

[0117]FIG. 90 shows the amino acid sequence (SEQ ID NO:90) derived fromthe coding sequence of SEQ ID NO:89 shown in FIG. 89.

[0118]FIG. 91 shows a nucleotide sequence (SEQ ID NO:91) of a nativesequence PRO1327 cDNA, wherein SEQ ID NO:91 is a clone designated hereinas “DNA66521-1583”.

[0119]FIG. 92 shows the amino acid sequence (SEQ ID NO:92) derived fromthe coding sequence of SEQ ID NO:91 shown in FIG. 91.

[0120]FIG. 93 shows a nucleotide sequence (SEQ ID NO:93) of a nativesequence PRO1328 cDNA, wherein SEQ ID NO:93 is a clone designated hereinas “DNA66658-1584”.

[0121]FIG. 94 shows the amino acid sequence (SEQ ID NO:94) derived fromthe coding sequence of SEQ ID NO:93 shown in FIG. 93.

[0122]FIG. 95 shows a nucleotide sequence (SEQ ID NO:95) of a nativesequence PRO1329 cDNA, wherein SEQ ID NO:95 is a clone designated hereinas “DNA66660-1585”.

[0123]FIG. 96 shows the amino acid sequence (SEQ ID NO:96) derived fromthe coding sequence of SEQ ID NO:95 shown in FIG. 95.

[0124]FIG. 97 shows a nucleotide sequence (SEQ ID NO:97) of a nativesequence PRO1340 cDNA, wherein SEQ ID NO:97 is a clone designated hereinas “DNA66663-1598”.

[0125]FIG. 98 shows the amino acid sequence (SEQ ID NO:98) derived fromthe coding sequence of SEQ ID NO:97 shown in FIG. 97.

[0126]FIG. 99 shows a nucleotide sequence (SEQ ID NO:99) of a nativesequence PRO1342 cDNA, wherein SEQ ID NO:99 is a clone designated hereinas “DNA66674-1599”.

[0127]FIG. 100 shows the amino acid sequence (SEQ ID NO:100) derivedfrom the coding sequence of SEQ ID NO:99 shown in FIG. 99.

[0128]FIG. 101 shows a nucleotide sequence (SEQ ID NO:101) of a nativesequence PRO3579 cDNA, wherein SEQ ID NO:101 is a clone designatedherein as “DNA68862-2546”.

[0129]FIG. 102 shows the amino acid sequence (SEQ ID NO:102) derivedfrom the coding sequence of SEQ ID NO:101 shown in FIG. 101.

[0130]FIG. 103 shows a nucleotide sequence (SEQ ID NO:103) of a nativesequence PRO1472 cDNA, wherein SEQ ID NO:103 is a clone designatedherein as “DNA68866-1644”.

[0131]FIG. 104 shows the amino acid sequence (SEQ ID NO:104) derivedfrom the coding sequence of SEQ ID NO:103 shown in FIG. 103.

[0132]FIG. 105 shows a nucleotide sequence (SEQ ID NO:105) of a nativesequence PRO1461 cDNA, wherein SEQ ID NO:105 is a clone designatedherein as “DNA68871-1638”.

[0133]FIG. 106 shows the amino acid sequence (SEQ ID NO:106) derivedfrom the coding sequence of SEQ ID NO:105 shown in FIG. 105.

[0134]FIG. 107 shows a nucleotide sequence (SEQ ID NO:107) of a nativesequence PRO1568 cDNA, wherein SEQ ID NO:107 is a clone designatedherein as “DNA68880-1676”.

[0135]FIG. 108 shows the amino acid sequence (SEQ ID NO:108) derivedfrom the coding sequence of SEQ ID NO:107 shown in FIG. 107.

[0136]FIG. 109 shows a nucleotide sequence (SEQ ID NO:109) of a nativesequence PRO1753 cDNA, wherein SEQ ID NO:109 is a clone designatedherein as “DNA68883-1691”.

[0137]FIG. 110 shows the amino acid sequence (SEQ ID NO:110) derivedfrom the coding sequence of SEQ ID NO:109 shown in FIG. 109.

[0138]FIG. 111 shows a nucleotide sequence (SEQ ID NO:111) of a nativesequence PRO1570 cDNA, wherein SEQ ID NO:11 is a clone designated hereinas “DNA68885-1678”.

[0139]FIG. 112 shows the amino acid sequence (SEQ ID NO:112) derivedfrom the coding sequence of SEQ ID NO:111 shown in FIG. 111.

[0140]FIG. 113 shows a nucleotide sequence (SEQ ID NO:113) of a nativesequence PRO1446 cDNA, wherein SEQ ID NO:113 is a clone designatedherein as “DNA71277-1636”.

[0141]FIG. 114 shows the amino acid sequence (SEQ ID NO:114) derivedfrom the coding sequence of SEQ ID NO:113 shown in FIG. 113.

[0142]FIG. 115 shows a nucleotide sequence (SEQ ID NO:115) of a nativesequence PRO1565 cDNA, wherein SEQ ID NO:115 is a clone designatedherein as “DNA73727-1673”.

[0143]FIG. 116 shows the amino acid sequence (SEQ ID NO:116) derivedfrom the coding sequence of SEQ ID NO: 15 shown in FIG. 115.

[0144]FIG. 117 shows a nucleotide sequence (SEQ ID NO: 17) of a nativesequence PRO1572 cDNA, wherein SEQ ID NO:117 is a clone designatedherein as “DNA73734-1680”.

[0145]FIG. 118 shows the amino acid sequence (SEQ ID NO:118) derivedfrom the coding sequence of SEQ ID NO:117 shown in FIG. 117.

[0146]FIG. 119 shows a nucleotide sequence (SEQ ID NO:119) of a nativesequence PRO1573 cDNA, wherein SEQ ID NO:119 is a clone designatedherein as “DNA73735-1681”.

[0147]FIG. 120 shows the amino acid sequence (SEQ ID NO:120) derivedfrom the coding sequence of SEQ ID NO: 19 shown in FIG. 119.

[0148]FIG. 121 shows a nucleotide sequence (SEQ ID NO: 121) of a nativesequence PRO1550 cDNA, wherein SEQ ID NO:121 is a clone designatedherein as “DNA76393-1664”.

[0149]FIG. 1222 shows the amino acid sequence (SEQ ID NO:122) derivedfrom the coding sequence of SEQ ID NO:121 shown in FIG. 121.

[0150]FIG. 123 shows a nucleotide sequence (SEQ ID NO:123) of a nativesequence PRO1693 cDNA, wherein SEQ ID NO:123 is a clone designatedherein as “DNA77301-1708”.

[0151]FIG. 124 shows the amino acid sequence (SEQ ID NO:124) derivedfrom the coding sequence of SEQ ID NO:123 shown in FIG. 123.

[0152]FIG. 125 shows a nucleotide sequence (SEQ ID NO:125) of a nativesequence PRO1566 cDNA, wherein SEQ ID NO:125 is a clone designatedherein as “DNA77568-1626”.

[0153]FIG. 126 shows the amino acid sequence (SEQ ID NO:126) derivedfrom the coding sequence of SEQ ID NO:125 shown in FIG. 125.

[0154]FIG. 127 shows a nucleotide sequence (SEQ ID NO:127) of a nativesequence PRO1774 cDNA, wherein SEQ ID NO:127 is a clone designatedherein as “DNA77626-1705”.

[0155]FIG. 128 shows the amino acid sequence (SEQ ID NO:128) derivedfrom the coding sequence of SEQ ID NO:127 shown in FIG. 127.

[0156]FIG. 129 shows a nucleotide sequence (SEQ ID NO:129) of a nativesequence PRO1928 cDNA, wherein SEQ ID NO:129 is a clone designatedherein as “DNA81754-2532”.

[0157]FIG. 130 shows the amino acid sequence (SEQ ID NO:130) derivedfrom the coding sequence of SEQ ID NO:129 shown in FIG. 129.

[0158]FIG. 131 shows a nucleotide sequence (SEQ ID NO:131) of a nativesequence PRO1865 cDNA, wherein SEQ ID NO:131 is a clone designatedherein as “DNA81757-2512”.

[0159]FIG. 132 shows the amino acid sequence (SEQ ID NO:132) derivedfrom the coding sequence of SEQ ID NO:131 shown in FIG. 131.

[0160]FIG. 133 shows a nucleotide sequence (SEQ ID NO:133) of a nativesequence PRO1925 cDNA, wherein SEQ ID NO:133 is a clone designatedherein as “DNA82302-2529”.

[0161]FIG. 134 shows the amino acid sequence (SEQ ID NO:134) derivedfrom the coding sequence of SEQ ID NO:133 shown in FIG. 133.

[0162]FIG. 135 shows a nucleotide sequence (SEQ ID NO:135) of a nativesequence PRO1926 cDNA, wherein SEQ ID NO:135 is a clone designatedherein as “DNA82340-2530”.

[0163]FIG. 136 shows the amino acid sequence (SEQ ID NO:136) derivedfrom the coding sequence of SEQ ID NO:135 shown in FIG. 135.

[0164]FIG. 137 shows a nucleotide sequence (SEQ ID NO:137) of a nativesequence PRO1801 cDNA, wherein SEQ ID NO:137 is a clone designatedherein as “DNA83500-2506”.

[0165]FIG. 138 shows the amino acid sequence (SEQ ID NO:138) derivedfrom the coding sequence of SEQ ID NO:137 shown in FIG. 137.

[0166]FIG. 139 shows a nucleotide sequence (SEQ ID NO:139) of a nativesequence PRO4405 cDNA, wherein SEQ ID NO:139 is a clone designatedherein as “DNA84920-2614”.

[0167]FIG. 140 shows the amino acid sequence (SEQ ID NO:140) derivedfrom the coding sequence of SEQ ID NO:139 shown in FIG. 139.

[0168]FIG. 141 shows a nucleotide sequence (SEQ ID NO:141) of a nativesequence PRO3435 cDNA, wherein SEQ iD NO:141 is a clone designatedherein as “DNA85066-2534”.

[0169]FIG. 142 shows the amino acid sequence (SEQ ID NO:142) derivedfrom the coding sequence of SEQ ID NO:141 shown in FIG. 141.

[0170]FIG. 143 shows a nucleotide sequence (SEQ ID NO:143) of a nativesequence PRO3543 cDNA, wherein SEQ ID NO:143 is a clone designatedherein as “DNA86571-2551”.

[0171]FIG. 144 shows the amino acid sequence (SEQ ID NO:144) derivedfrom the coding sequence of SEQ ID NO:143 shown in FIG. 143.

[0172]FIG. 145 shows a nucleotide sequence (SEQ ID NO:145) of a nativesequence PRO3443 cDNA, wherein SEQ ID NO:145 is a clone designatedherein as “DNA87991-2540”.

[0173]FIG. 146 shows the amino acid sequence (SEQ ID NO:146) derivedfrom the coding sequence of SEQ ID NO:145 shown in FIG. 145.

[0174]FIG. 147 shows a nucleotide sequence (SEQ ID NO:147) of a nativesequence PRO3442 cDNA, wherein SEQ ID NO:147 is a clone designatedherein as “DNA92238-2539”.

[0175]FIG. 148 shows the amino acid sequence (SEQ ID NO:148) derivedfrom the coding sequence of SEQ ID NO:147 shown in FIG. 147.

[0176]FIG. 149 shows a nucleotide sequence (SEQ ID NO:149) of a nativesequence PRO5990 cDNA, wherein SEQ ID NO:149 is a clone designatedherein as “DNA96042-2682”.

[0177]FIG. 150 shows the amino acid sequence (SEQ ID NO:150) derivedfrom the coding sequence of SEQ ID NO:149 shown in FIG. 149.

[0178]FIG. 151 shows a nucleotide sequence (SEQ ID NO:151) of a nativesequence PRO4342 cDNA, wherein SEQ ID NO:151 is a clone designatedherein as “DNA96787-2534”.

[0179]FIG. 152 shows the amino acid sequence (SEQ ID NO:152) derivedfrom the coding sequence of SEQ ID NO:151 shown in FIG. 151.

[0180]FIG. 153 shows a nucleotide sequence (SEQ ID NO:153) of a nativesequence PRO10096 cDNA, wherein SEQ ID NO:153 is a clone designatedherein as “DNA125185-2806”.

[0181]FIG. 154 shows the amino acid sequence (SEQ ID NO:154) derivedfrom the coding sequence of SEQ ID NO:153 shown in FIG. 153.

[0182]FIG. 155) shows a nucleotide sequence (SEQ ID NO:155) of a nativesequence PRO10272 cDNA, wherein SEQ ID NO:155 is a clone designatedherein as “DNA147531-2821”.

[0183]FIG. 156 shows the amino acid sequence (SEQ ID NO:156) derivedfrom the coding sequence of SEQ ID NO:155 shown in FIG. 155.

[0184]FIG. 157 shows a nucleotide sequence (SEQ ID NO:157) of a nativesequence PRO5801 cDNA, wherein SEQ ID NO:157 is a clone designatedherein as “DNA115291-2681”.

[0185]FIG. 158 shows the amino acid sequence (SEQ ID NO:158) derivedfrom the coding sequence of SEQ ID NO:157 shown in FIG. 157.

[0186]FIG. 159 shows a nucleotide sequence (SEQ ID NO:159) of a nativesequence PRO20110 cDNA, wherein SEQ ID NO:159 is a clone designatedherein as “DNA166819”.

[0187]FIG. 160 shows the amino acid sequence (SEQ ID NO:160) derivedfrom the coding sequence of SEQ ID NO:159 shown in FIG. 159.

[0188]FIG. 161 shows a nucleotide sequence (SEQ ID NO:161) of a nativesequence PRO20040 cDNA, wherein SEQ ID NO:161 is a clone designatedherein as “DNA164625′-2890”.

[0189]FIG. 162 shows the amino acid sequence (SEQ ID NO: 162) derivedfrom the coding sequence of SEQ ID NO:161 shown in FIG. 161.

[0190]FIG. 163 shows a nucleotide sequence (SEQ ID NO: 163) of a nativesequence PRO20233 cDNA, wherein SEQ ID NO:163 is a clone designatedherein as “DNA1656085”.

[0191]FIG. 164 shows the amino acid sequence (SEQ ID NO:164) derivedfrom the coding sequence of SEQ ID NO:163 shown in FIG. 163.

[0192]FIG. 165 shows a nucleotide sequence (SEQ ID NO: 65) of a nativesequence PRO19670 cDNA, wherein SEQ ID NO:165 is a clone designatedherein as “DNA131639-2874”.

[0193]FIG. 166 shows the amino acid sequence (SEQ ID NO:166) derivedfrom the coding sequence of SEQ ID NO:165 shown in FIG. 165.

[0194]FIG. 167 shows a nucleotide sequence (SEQ ID NO:167) of a nativesequence PRO1890 cDNA, wherein SEQ ID NO:167 is a clone designatedherein as “DNA79230-2525”.

[0195]FIG. 168 shows the amino acid sequence (SEQ ID NO:168) derivedfrom the coding sequence of SEQ ID NO:167 shown in FIG. 167.

DETAILED DESCRIPTION

[0196] I. Definitions

[0197] The terms “PRO polypeptide” and “PRO” as used herein and whenimmediately followed by a numerical designation refer to variouspolypeptides, wherein the complete designation (i.e., PRO/number) refersto specific polypeptide sequences as described herein. The terms“PRO/number polypeptide” and “PRO/number” wherein the term “number” isprovided as an actual numerical designation as used herein encompassnative sequence polypeptides and polypeptide variants (which are furtherdefined herein). The PRO polypeptides described herein may be isolatedfrom a variety of sources, such as from human tissue types or fromanother source, or prepared by recombinant or synthetic methods. Theterm “PRO polypeptide” refers to each individual PRO/number polypeptidedisclosed herein. All disclosures in this specification which refer tothe “PRO polypeptide” refer to each of the polypeptides individually aswell as jointly. For example, descriptions of the preparation of,purification of, derivation of, formation of antibodies to or against,administration of, compositions containing, treatment of a disease with,etc., pertain to each polypeptide of the invention individually. Theterm “PRO polypeptide” also includes variants of the PRO/numberpolypeptides disclosed herein.

[0198] A “native sequence PRO polypeptide” comprises a polypeptidehaving the same amino acid sequence as the corresponding PRO polypeptidederived from nature. Such native sequence PRO polypeptides can beisolated from nature or can be produced by recombinant or syntheticmeans. The term “native sequence PRO polypeptide” specificallyencompasses naturally-occurring truncated or secreted forms of thespecific PRO, polypeptide (e.g., an extracellular domain sequence),naturally-occurring variant forms (e.g., alternatively spliced forms)and naturally-occurring allelic variants of the polypeptide. In variousembodiments of the invention, the native sequence PRO polypeptidesdisclosed herein are mature or full-length native sequence polypeptidescomprising the full-length amino acids sequences shown in theaccompanying figures. Start and stop codons are shown in bold font andunderlined in the figures. However, while the PRO polypeptide disclosedin the accompanying figures are shown to begin with methionine residuesdesignated herein as amino acid position 1 in the figures, it isconceivable and possible that other methionine residues located eitherupstream or downstream from the amino acid position 1 in the figures maybe employed as the starting amino acid residue for the PRO polypeptides.

[0199] The PRO polypeptide “extracellular domain” or “ECD” refers to aform of the PRO polypeptide which is essentially free of thetransmembrane and cytoplasmic domains. Ordinarily, a PRO polypeptide ECDwill have less than 1% of such transmembrane and/or cytoplasmic domainsand preferably, will have less than 0.5% of such domains. It will beunderstood that any transmembrane domains identified for the PROpolypeptides of the present invention are identified pursuant tocriteria routinely employed in the art for identifying that type ofhydrophobic domain. The exact boundaries of a transmembrane domain mayvary but most likely by no more than about 5 amino acids at either endof the domain as initially identified herein. Optionally, therefore, anextracellular domain of a PRO polypeptide may contain from about 5 orfewer amino acids on either side of the transmembranedomain/extracellular domain boundary as identified in the Examples orspecification and such polypeptides, with or without the associatedsignal peptide, and nucleic acid encoding them, are comtemplated by thepresent invention.

[0200] The approximate location of the “signal peptides” of the variousPRO polypeptides disclosed herein are shown in the present specificationand/or the accompanying figures. It is noted, however, that theC-terminal boundary of a signal peptide may vary, but most likely by nomore than about 5 amino acids on either side of the signal peptideC-terminal boundary as initially identified herein, wherein theC-terminal boundary of the signal peptide may be identified pursuant tocriteria routinely employed in the art for identifying that type ofamino acid sequence element (e.g., Nielsen et al., Prot. Eng. 10:1-6(1997) and von Heinje et al., Nucl. Acids. Res. 14:4683-4690 (1986)).Moreover, it is also recognized that, in some cases, cleavage of asignal sequence from a secreted polypeptide is not entirely uniform,resulting in more than one secreted species. These mature polypeptides,where the signal peptide is cleaved within no more than about 5 aminoacids on either side of the C-terminal boundary of the signal peptide asidentified herein, and the polynucleotides encoding them, arecontemplated by the present invention.

[0201] “PRO polypeptide variant” means an active PRO polypeptide asdefined above or below having at least about 80% amino acid sequenceidentity with a full-length native sequence PRO polypeptide sequence asdisclosed herein, a PRO polypeptide sequence lacking the signal peptideas disclosed herein, an extracellular domain of a PRO polypeptide, withor without the signal peptide, as disclosed herein or any other fragmentof a full-length PRO polypeptide sequence as disclosed herein. Such PROpolypeptide variants include, for instance, PRO polypeptides wherein oneor more amino acid residues are added, or deleted, at the N- orC-terminus of the full-length native amino acid sequence. Ordinarily, aPRO polypeptide variant will have at least about 80% amino acid sequenceidentity, alternatively at least about 81% amino acid sequence identity,alternatively at least about 82% amino acid sequence identity,alternatively at least about 83% amino acid sequence identity,alternatively at least about 84%5 amino acid sequence identity,alternatively at least about 85% amino acid sequence identity,alternatively at least about 86% amino acid sequence identity,alternatively at least about 87% amino acid sequence identity,alternatively at least about 88%i amino acid sequence identity,alternatively at least about 89% amino acid sequence identity,alternatively at least about 90% amino acid sequence identity,alternatively at least about 91% amino acid sequence identity,alternatively at least about 92%, amino acid sequence identity,alternatively at least about 93% amino acid sequence identity,alternatively at least about 94% amino acid sequence identity,alternatively at least about 95% amino acid sequence identity,alternatively at least about 9696 amino acid sequence identity,alternatively at least about 97% amino acid sequence identity,alternatively at least about 98% amino acid sequence identity andalternatively at least about 99% amino acid sequence identity to afull-length native sequence PRO polypeptide sequence as disclosedherein, a PRO polypeptide sequence lacking the signal peptide asdisclosed herein, an extracellular domain of a PRO polypeptide, with orwithout the signal peptide, as disclosed herein or any otherspecifically defined fragment of a full-length PRO polypeptide sequenceas disclosed herein. Ordinarily, PRO variant polypeptides are at leastabout 10 amino acids in length, alternatively at least about 20 aminoacids in length, alternatively at least about 30 amino acids in length,alternatively at least about 40 amino acids in length, alternatively atleast about 50 amino acids in length, alternatively at least about 60amino acids in length, alternatively at least about 70 amino acids inlength, alternatively at least about 80 amino acids in length,alternatively at least about 90 amino acids in length, alternatively atleast about 100 amino acids in length, alternatively at least about 150amino acids in length, alternatively at least about 200 amino acids inlength, alternatively at least about 300 amino acids in length, or more.

[0202] “Percent (%) amino acid sequence identity” with respect to thePRO polypeptide sequences identified herein is defined as the percentageof amino acid residues in a candidate sequence that are identical withthe amino acid residues in the specific PRO polypeptide sequence, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity, and not considering anyconservative substitutions as part of the sequence identity. Alignmentfor purposes of determining percent amino acid sequence identity can beachieved in various ways that are within the skill in the art, forinstance, using publicly available computer software such as BLAST,BLAST-2, ALIGN or Megalign (DNASTAR) software. Those skilled in the artcan determine appropriate parameters for measuring alignment, includingany algorithms needed to achieve maximal alignment over the full lengthof the sequences being compared. For purposes herein, however, % aminoacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0203] In situations where ALIGN-2 is employed for amino acid sequencecomparisons, the % amino acid sequence identity of a given amino acidsequence A to, with, or against a given amino acid sequence B (which canalternatively be phrased as a given amino acid sequence A that has orcomprises a certain % amino acid sequence identity to, with, or againsta given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0204] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program ALIGN-2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A. As examples of % amino acid sequenceidentity calculations using this method, Tables 2 and 3 demonstrate howto calculate the % amino acid sequence identity of the amino acidsequence designated “Comparison Protein” to the amino acid sequencedesignated “PRO”, wherein “PRO” represents the amino acid sequence of ahypothetical PRO polypeptide of interest, “Comparison Protein”represents the amino acid sequence of a polypeptide against which the“PRO” polypeptide of interest is being compared, and “X”, “Y” and “Z”each represent different hypothetical amino acid residues.

[0205] Unless; specifically stated otherwise, all % amino acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-2 computer program. However, % aminoacid sequence identity values may also be obtained as described below byusing the WU-BLAST-2 computer program (Altschul et al., Methods inEnzymology 266:460-480 (1996)). Most of the WU-BLAST-2 search parametersare set to the default values. Those not set to default values, i.e.,the adjustable parameters, are set with the following values: overlapspan=1, overlap fraction=0.125, word threshold (T)=11, and scoringmatrix=BLOSUM62. When WU-BLAST-2 is employed, a % amino acid sequenceidentity value is determined by dividing (a) the number of matchingidentical amino acid residues between the amino acid sequence of the PROpolypeptide of interest having a sequence derived from the native PROpolypeptide and the comparison amino acid sequence of interest (i.e.,the sequence against which the PRO polypeptide of interest is beingcompared which may be a PRO variant polypeptide) as determined byWU-BLAST-2 by (b) the total number of amino acid residues of the PROpolypeptide of interest. For example, in the statement “a polypeptidecomprising an the amino acid sequence A which has or having at least 80%amino acid sequence identity to the amino acid sequence B”, the aminoacid sequence A is the comparison amino acid sequence of interest andthe amino acid sequence B is the amino acid sequence of the PROpolypeptide of interest.

[0206] Percent amino acid sequence identity may also be determined usingthe sequence comparison program NCBI-BLAST2 (Altschul et al., NucleicAcids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequence comparisonprogram may be downloaded from http://wwvv.ncbi.nim.nih.gov or otherwiseobtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0207] In situations where NCBI-BLAST2 is employed for amino acidsequence comparisons, the % amino acid sequence identity of a givenamino acid sequence A to, with, or against a given amino acid sequence B(which can alternatively be phrased as a given amino acid sequence Athat has or comprises a certain % amino acid sequence identity to, with,or against a given amino acid sequence B) is calculated as follows:

100 times the fraction X/Y

[0208] where X is the number of amino acid residues scored as identicalmatches by the sequence alignment program NCBI-BLAST2 in that program'salignment of A and B, and where Y is the total number of amino acidresidues in B. It will be appreciated that where the length of aminoacid sequence A is not equal to the length of amino acid sequence B, the% amino acid sequence identity of A to B will not equal the % amino acidsequence identity of B to A.

[0209] “PRO variant polynucleotide” or “PRO variant nucleic acidsequence” means a nucleic acid molecule which encodes an active PROpolypeptide as defined below and which has at least about 80% nucleicacid sequence identity with a nucleotide acid sequence encoding afull-length native sequence PRO polypeptide sequence as disclosedherein, a full-length native sequence PRO polypeptide sequence lackingthe signal peptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal peptide, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Ordinarily, a PRO variant polynucleotide will have atleast about 80% nucleic acid sequence identity, alternatively at leastabout 81% nucleic acid sequence identity, alternatively at least about82% nucleic acid sequence identity, alternatively at least about 83%nucleic acid sequence identity, alternatively at least about 84% nucleicacid sequence identity, alternatively at least about 85% nucleic acidsequence identity, alternatively at least about 86% nucleic acidsequence identity, alternatively at least about 87% nucleic acidsequence identity, alternatively at least about 88% nucleic acidsequence identity, alternatively at least about 89% nucleic acidsequence identity, alternatively at least about 90% nucleic acidsequence identity, alternatively at least about 91% nucleic acidsequence identity, alternatively at least about 92% nucleic acidsequence identity, alternatively at least about 93% nucleic acidsequence identity, alternatively at least about 94% nucleic acidsequence identity, alternatively at least about 95% nucleic acidsequence identity, alternatively at least about 96% nucleic acidsequence identity, alternatively at least about 97% nucleic acidsequence identity, alternatively at least about 98%, nucleic acidsequence identity and alternatively at least about 99% nucleic acidsequence identity with a nucleic acid sequence encoding a full-lengthnative sequence PRO polypeptide sequence as disclosed herein, afull-length native sequence PRO polypeptide sequence lacking the signalpeptide as disclosed herein, an extracellular domain of a PROpolypeptide, with or without the signal sequence, as disclosed herein orany other fragment of a full-length PRO polypeptide sequence asdisclosed herein. Variants do not encompass the native nucleotidesequence.

[0210] Ordinarily, PRO variant polynucleotides are at least about 30nucleotides in length, alternatively at least about 60 nucleotides inlength, alternatively at least about 90 nucleotides in length,alternatively at least about 120 nucleotides in length, alternatively atleast about 150 nucleotides in length, alternatively at least about 180nucleotides in length, alternatively at least about 210 nucleotides inlength, alternatively at least about 240 nucleotides in length,alternatively at least about 270 nucleotides in length, alternatively atleast about 300 nucleotides in length, alternatively at least about 450nucleotides in length, alternatively at least about 600 nucleotides inlength, alternatively at least about 900 nucleotides in length, or more.

[0211] “Percent (%) nucleic acid sequence identity” with respect toPRO-encoding nucleic acid sequences identified herein is defined as thepercentage of nucleotides in a candidate sequence that are identicalwith the nucleotides in the PRO nucleic acid sequence of interest, afteraligning the sequences and introducing gaps, if necessary, to achievethe maximum percent sequence identity. Alignment for purposes ofdetermining percent nucleic acid sequence identity can be achieved invarious ways that are within the skill in the art, for instance, usingpublicly available computer software such as BLAST, BLAST-2, ALIGN orMegalign (DNASTAR) software. For purposes herein, however, % nucleicacid sequence identity values are generated using the sequencecomparison computer program ALIGN-2, wherein the complete source codefor the ALIGN-2 program is provided in Table 1 below. The ALIGN-2sequence comparison computer program was authored by Genentech, Inc. andthe source code shown in Table 1 below has been filed with userdocumentation in the U.S. Copyright Office, Washington D.C., 20559,where it is registered under U.S. Copyright Registration No. TXU510087.The ALIGN-2 program is publicly available through Genentech, Inc., SouthSan Francisco, Calif. or may be compiled from the source code providedin Table 1 below. The ALIGN-2 program should be compiled for use on aUNIX operating system, preferably digital UNIX V4.0D. All sequencecomparison parameters are set by the ALIGN-2 program and do not vary.

[0212] In situations where ALIGN-2 is employed for nucleic acid sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0213] where W is the number of nucleotides scored as identical matchesby the sequence alignment program ALIGN-2 in that program's alignment ofC and D, and where Z is the total number of nucleotides in D. It will beappreciated that where the length of nucleic acid sequence C is notequal to the length of nucleic acid sequence D, the % nucleic acidsequence identity of C to D will not equal the % nucleic acid sequenceidentity of D to C. As examples of % nucleic acid sequence identitycalculations, Tables 4 and 5, demonstrate how to calculate the % nucleicacid sequence identity of the nucleic acid sequence designated“Comparison DNA” to the nucleic acid sequence designated “PRO-DNA”,wherein “PRO-DNA” represents a hypothetical PRO-encoding nucleic acidsequence of interest, “Comparison DNA” represents the nucleotidesequence of a nucleic acid molecule against which the “PRO-DNA” nucleicacid molecule of interest is being compared, and “N”, “L” and “V” eachrepresent different hypothetical nucleotides.

[0214] Unless specifically stated otherwise, all % nucleic acid sequenceidentity values used herein are obtained as described in the immediatelypreceding paragraph using the ALIGN-0.2 computer program. However, %nucleic acid sequence identity values may also be obtained as describedbelow by using the WU-BLAST-2 computer program (Altschul et al., Methodsin Enzymology 266:460-480 (1996)). Most of the WU-BLAST-2 searchparameters are set to the default values. Those not set to defaultvalues, i.e., the adjustable parameters, are set with the followingvalues: overlap span=1, overlap fraction=0.125, word threshold (T)=11,and scoring matrix=BLOSUM62. When WU-BLAST-2 is employed, a % nucleicacid sequence identity value is determined by dividing (a) the number ofmatching identical nucleotides between the nucleic acid sequence of thePRO polypeptide-encoding nucleic acid molecule of interest having asequence derived from the native sequence PRO polypeptide-encodingnucleic acid and the comparison nucleic acid molecule of interest (i.e.,the sequence against which the PRO polypeptide-encoding nucleic acidmolecule of interest is being compared which may be a variant PROpolynucleotide) as determined by WU-BLAST-2 by (b) the total number ofnucleotides of the PRO polypeptide-encoding nucleic acid molecule ofinterest. For example, in the statement “an isolated nucleic acidmolecule comprising a nucleic acid sequence A which has. or having atleast 80% nucleic acid sequence identity to the nucleic acid sequenceB”, the nucleic acid sequence A is the comparison nucleic acid moleculeof interest and the nucleic acid sequence B is the nucleic acid sequenceof the PRO polypeptide-encoding nucleic acid molecule of interest.

[0215] Percent nucleic acid sequence identity may also be determinedusing the sequence comparison program NCBI-BLAST2 (Altschul et al.,Nucleic Acids Res. 25:3389-3402 (1997)). The NCBI-BLAST2 sequencecomparison program may be downloaded from http://www.ncbi.nim.nih.gov orotherwise obtained from the National Institute of Health, Bethesda, Md.NCBI-BLAST2 uses several search parameters, wherein all of those searchparameters are set to default values including, for example, unmask=yes,strand=all, expected occurrences=10, minimum low complexity length=15/5,multi-pass e-value=0.01, constant for multi-pass=25, dropoff for finalgapped alignment=25 and scoring matrix=BLOSUM62.

[0216] In situations where NCBI-BLAST2 is employed for sequencecomparisons, the % nucleic acid sequence identity of a given nucleicacid sequence C to, with, or against a given nucleic acid sequence D(which can alternatively be phrased as a given nucleic acid sequence Cthat has or comprises a certain % nucleic acid sequence identity to,with, or against a given nucleic acid sequence D) is calculated asfollows:

100 times the fraction W/Z

[0217] where W is the number of nucleotides scored as identical matchesby the sequence alignment program NCBI-BLAST2 in that program'salignment of C and D, and where Z is the total number of nucleotides inD. It will be appreciated that where the length of nucleic acid sequenceC is not equal to the length of nucleic acid sequence D, the % nucleicacid sequence identity of C to D will not equal the % nucleic acidsequence identity of D to C.

[0218] In other embodiments, PRO variant polynucleotides are nucleicacid molecules that encode an active PRO polypeptide and which arecapable of hybridizing, preferably under stringent hybridization andwash conditions, to nucleotide sequences encoding a full-length PROpolypeptide as disclosed herein. PRO variant polypeptides may be thosethat are encoded by a PRO variant polynucleotide.

[0219] “Isolated,” when used to describe the various polypeptidesdisclosed herein, means polypeptide that has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials thatwould typically interfere with diagnostic or therapeutic uses for thepolypeptide, and may include enzymes, hormones, and other proteinaceousor non-proteinaceous solutes. In preferred embodiments, the polypeptidewill be purified (1) to a degree sufficient to obtain at least 15residues of N-terminal or internal amino acid sequence by use of aspinning cup sequenator, or (2) to homogeneity by SDS-PAGE undernon-reducing or reducing conditions using Coomassie blue or, preferably,silver stain. Isolated polypeptide includes polypeptide in situ withinrecombinant cells, since at least one component of the PRO polypeptidenatural environment will not be present. Ordinarily, however, isolatedpolypeptide will be prepared by at least one purification step.

[0220] An “isolated” PRO polypeptide-encoding nucleic acid or otherpolypeptide-encoding nucleic acid is a nucleic acid molecule that isidentified and separated from at least one contaminant nucleic acidmolecule with which it is ordinarily associated in the natural source ofthe polypeptide-encoding nucleic acid. An isolated polypeptide-encodingnucleic acid molecule is other than in the form or setting in which itis found in nature. Isolated polypeptide-encoding nucleic acid moleculestherefore are distinguished from the specific polypeptide-encodingnucleic acid molecule as it exists in natural cells. However, anisolated polypeptide-encoding nucleic acid molecule includespolypeptide-encoding nucleic acid molecules contained in cells thatordinarily express the polypeptide where, for example, the nucleic acidmolecule is in a chromosomal location different from that of naturalcells.

[0221] The term “control sequences” refers to DNA sequences necessaryfor the expression of an operably linked coding sequence in a particularhost organism. The control sequences that are suitable for prokaryotes,for example, include a promoter, optionally an operator sequence, and aribosome binding site. Eukaryotic cells are known to utilize promoters,polyadenylation signals, and enhancers.

[0222] Nucleic acid is “operably linked” when it is placed into afunctional relationship with another nucleic acid sequence. For example,DNA for a presequence or secretory leader is operably linked to DNA fora polypeptide if it is expressed as a preprotein that participates inthe secretion of the polypeptide; a promoter or enhancer is operablylinked to a coding sequence if it affects the transcription of thesequence; or a ribosome binding site is operably linked to a codingsequence if it is positioned so as to facilitate translation. Generally,“operably linked” means that the DNA sequences being linked arecontiguous, and, in the case of a secretory leader, contiguous and inreading phase. However, enhancers do not have to be contiguous. Linkingis accomplished by ligation at convenient restriction sites. If suchsites do not exist, the synthetic oligonucleotide adaptors or linkersare used in accordance with conventional practice.

[0223] The term “antibody” is used in the broadest sense andspecifically covers, for example, single anti-PRO monoclonal antibodies(including agonist, antagonist, and neutralizing antibodies), anti-PROantibody compositions with polyepitopic specificity, single chainanti-PRO antibodies, and fragments of anti-PRO antibodies (see below).The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally-occurring mutations that may be present inminor amounts.

[0224] “Stringency” of hybridization reactions is readily determinableby one of ordinary skill in the art, and generally is an empiricalcalculation dependent upon probe length, washing temperature, and saltconcentration. In general, longer probes require higher temperatures forproper annealing, while shorter probes need lower temperatures.Hybridization generally depends on the ability of denatured DNA toreanneal when complementary strands are present in an environment belowtheir melting temperature. The higher the degree of desired homologybetween the probe and hybridizable sequence, the higher the relativetemperature which can be used. As a result, it follows that higherrelative temperatures would tend to make the reaction conditions morestringent, while lower temperatures less so. For additional details andexplanation of stringency of hybridization reactions, see Ausubel etal., Current Protocols in Molecular Biology, Wiley IntersciencePublishers, (1995).

[0225] “Stringent conditions” or “high stringency conditions”, asdefined herein, may be identified by those that: (1) employ low ionicstrength and high temperature for washing, for example 0.015 M sodiumchloride/0.0015 M sodium citrate/0.1% sodium dodecyl sulfate at 50° C.;(2) employ during hybridization a denaturing agent, such as formamide,for example, 50% (v/v) formamide with 0.1% bovine serum albumin/0.1%Ficoll/0.1% polyvinylpyrrolidone/50 mM sodium phosphate buffer at pH 6.5with 750 mM sodium chloride, 75 mM sodium citrate at 42° C.; or (3)employ 50% formamide, 5× SSC (0.75 M NaCl, 0.075 M sodium citrate), 50mM sodium phosphate (pH 6.8), 0.1% sodium pyrophosphate, 5× Denhardt'ssolution, sonicated salmon sperm DNA (50 μg/ml), 0.1% SDS, and 10%dextran sulfate at 42° C., with washes at 42° C. in 0.2× SSC (sodiumchloride/sodium citrate) and 50% formamide at 55° C., followed by ahigh-stringency wash consisting of 0.1× SSC containing EDTA at 55° C.

[0226] “Moderately stringent conditions” may be identified as describedby Sambrook et al., Molecular Cloning: A Laboratory Manual, New York:Cold Spring Harbor Press, 1989, and include the use of washing solutionand hybridization conditions (e.g., temperature, ionic strength and%SDS) less stringent that those described above. An example ofmoderately stringent conditions is overnight incubation at 37° C. in asolution comprising: 20% formamide, 5× SSC (150 mM NaCl, 15 mM trisodiumcitrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10%dextran sulfate, and 20 mg/ml denatured sheared salmon sperm DNA,followed by washing the filters in 1× SSC at about 37-50° C. The skilledartisan will recognize how to adjust the temperature, ionic strength,etc. as necessary to accommodate factors such as probe length and thelike.

[0227] The term “epitope tagged” when used herein refers to a chimericpolypeptide comprising a PRO polypeptide fused to a “tag polypeptide”.The tag polypeptide has enough residues to provide an epitope againstwhich an antibody can be made, yet is short enough such that it does notinterfere with activity of the polypeptide to which it is fused. The tagpolypeptide preferably also is fairly unique so that the antibody doesnot substantially cross-react with other epitopes. Suitable tagpolypeptides generally have at least six amino acid residues and usuallybetween about 8 and 50 amino acid residues (preferably, between about 10and 20 amino acid residues).

[0228] As used herein, the term “immunoadhesin” designates antibody-likemolecules which combine the binding specificity of a heterologousprotein (an “adhesin”) with the effector functions of immunoglobulinconstant domains. Structurally, the immunoadhesins comprise a fusion ofan amino acid sequence with the desired binding specificity which isother than the antigen recognition and binding site of an antibody(i.e., is “heterologous”), and an immunoglobulin constant domainsequence.

[0229] The adhesin part of an immunoadhesin molecule typically is acontiguous amino acid sequence comprising at least the binding site of areceptor or a ligand. The immunoglobulin constant domain sequence in theimmunoadhesin may be obtained from any immunoglobulin, such as IgG-1,IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE,IgD or IgM.

[0230] “Active” or “activity” for the purposes herein refers to form(s)of a PRO polypeptide which retain a biological and/or an immunologicalactivity of native or naturally-occurring PRO, wherein “biological”activity refers to a biological function (either inhibitory orstimulatory) caused by a native or naturally-occurring PRO other thanthe ability to induce the production of an antibody against an antigenicepitope possessed by a native or naturally-occurring PRO and an“immunological” activity refers to the ability to induce the productionof an antibody against an antigenic epitope possessed by a native ornaturally-occurring PRO.

[0231] The term “antagonist” is used in the broadest sense, and includesany molecule that partially or fully blocks, inhibits, or neutralizes abiological activity of a native PRO polypeptide disclosed herein. In asimilar manner, the term “agonist” is used in the broadest sense andincludes any molecule that mimics a biological activity of a native PROpolypeptide disclosed herein. Suitable agonist or antagonist moleculesspecifically include agonist or antagonist antibodies or antibodyfragments, fragments or amino acid sequence variants of native PROpolypeptides, peptides, antisense oligonucleotides, small organicmolecules, etc. Methods for identifying agonists or antagonists of a PROpolypeptide may comprise contacting a PRO polypeptide with a candidateagonist or antagonist molecule and measuring a detectable change in oneor more biological activities normally associated with the PROpolypeptide.

[0232] “Treatment” refers to both therapeutic treatment and prophylacticor preventative measures, wherein the object is to prevent or slow down(lessen) the targeted pathologic condition or disorder. Those in need oftreatment include those already with the disorder as well as those proneto have the disorder or those in whom the disorder is to be prevented.

[0233] “Chronic” administration refers to administration of the agent(s)in a continuous mode as opposed to an acute mode, so as to maintain theinitial therapeutic effect (activity) for an extended period of time.“Intermittent” administration is treatment that is not consecutivelydone without interruption, but rather is cyclic in nature.

[0234] “Mammal” for purposes of treatment refers to any animalclassified as a mammal, including humans, domestic and farm animals, andzoo, sports, or pet animals, such as dogs, cats, cattle, horses, sheep,pigs, goats, rabbits, etc. Preferably, the mammal is human.

[0235] Administration “in combination with” one or more furthertherapeutic agents includes simultaneous (concurrent) and consecutiveadministration in any order.

[0236] “Carriers” as used herein include pharmaceutically acceptablecarriers, excipients, or stabilizers which are nontoxic to the cell ormammal being exposed thereto at the dosages and concentrations employed.Often the physiologically acceptable carrier is an aqueous pH bufferedsolution. Examples of physiologically acceptable carriers includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid; low molecular weight (less thanabout 10 residues) polypeptide; proteins, such as serum albumin,gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, arginine or lysine; monosaccharides, disaccharides, andother carbohydrates including glucose, mannose, or dextrins; chelatingagents such as EDTA; sugar alcohols such as mannitol or sorbitol;salt-forming counterions such as sodium; and/or nonionic surfactantssuch as TWEEN™, polyethylene glycol (PEG), and PLURONIC™.

[0237] “Antibody fragments” comprise a portion of an intact antibody,preferably the antigen binding or variable region of the intactantibody. Examples of antibody fragments include Fab, Fab′, F(ab′)₂, andFv fragments; diabodies; linear antibodies (Zapata et al., Protein Eng.8(10): 1057-1062[1995]); single-chain antibody molecules; andmultispecific antibodies formed from antibody fragments.

[0238] Papain digestion of antibodies produces two identicalantigen-binding fragments, called “Fab” fragments, each with a singleantigen-binding site, and a residual “Fc” fragment, a designationreflecting the ability to crystallize readily. Pepsin treatment yieldsan F(ab′)₂ fragment that has two antigen-combining sites and is stillcapable of cross-linking antigen.

[0239] “Fv” is the minimum antibody fragment which contains a completeantigen-recognition and -binding site. This region consists of a dimerof one heavy- and one light-chain variable domain in tight, non-covalentassociation. It is in this configuration that the three CDRs of eachvariable domain interact to define an antigen-binding site on thesurface of the V_(H)-V_(L) dimer. Collectively, the six CDRs conferantigen-binding specificity to the antibody. However, even a singlevariable domain (or half of an Fv comprising only three CDRs specificfor an antigen) has the ability to recognize and bind antigen, althoughat a lower affinity than the entire binding site.

[0240] The Fab fragment also contains the constant domain of the lightchain and the first constant domain (CH1) of the heavy chain. Fabfragments differ from Fab′ fragments by the addition of a few residuesat the carboxy terminus of the heavy chain CH1 domain including one ormore cysteines from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

[0241] The “light chains” of antibodies (immunoglobulins) from anyvertebrate species can be assigned to one of two clearly distinct types,called kappa and lambda, based on the amino acid sequences of theirconstant domains.

[0242] Depending on the amino acid sequence of the constant domain oftheir heavy chains, immunoglobulins can be assigned to differentclasses. There are five major classes of immunoglobulins: IgA, IgD, IgE,IgG, and IgM, and several of these may be further divided intosubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2.“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of antibody, wherein these domains are present in a singlepolypeptide chain.

[0243] Preferably, the Fv polypeptide further comprises a polypeptidelinker between the V_(H) and V_(L) domains which enables the sFv to formthe desired structure for antigen binding. For a review of sFv, seePluckthun in The Pharmacology of Monoclonal Antibodies, vol. 113,Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0244] The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy-chain variabledomain (V_(H)) connected to a light-chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993,).

[0245] An “isolated” antibody is one which has been identified andseparated and/or recovered from a component of its natural environment.Contaminant components of its natural environment are materials whichwould interfere with diagnostic or therapeutic uses for the antibody,and may include enzymes, hormones, and other proteinaceous ornonproteinaceous solutes. In preferred embodiments, the antibody will bepurified (1) to greater than 95% by weight of antibody as determined bythe Lowry method, and most preferably more than 99% by weight, (2) to adegree sufficient to obtain at least 15 residues of N-terminal orinternal amino acid sequence by use of a spinning cup sequenator, or (3)to homogeneity by SDS-PAGE under reducing or nonreducing conditionsusing Coomassie blue or, preferably, silver stain. Isolated antibodyincludes the antibody in situ within recombinant cells since at leastone component of the antibody's natural environment will not be present.Ordinarily, however, isolated antibody will be prepared by at least onepurification step.

[0246] An antibody that “specifically binds to” or is “specific for” aparticular polypeptide or an epitope on a particular polypeptide is onethat binds to that particular polypeptide or epitope on a particularpolypeptide without substantially binding to any other polypeptide orpolypeptide epitope.

[0247] The word “label” when used herein refers to a detectable compoundor composition which is conjugated directly or indirectly to theantibody so as to generate a “labeled” antibody. The label may bedetectable by itself (e.g. radioisotope labels or fluorescent labels)or, in the case of an enzymatic label, may catalyze chemical alterationof a substrate compound or composition which is detectable.

[0248] By “solid phase” is meant a non-aqueous matrix to which theantibody of the present invention can adhere. Examples of solid phasesencompassed herein include those formed partially or entirely of glass(e.g., controlled pore glass), polysaccharides (e.g., agarose),polyacrylamides, polystyrene, polyvinyl alcohol and silicones. Incertain embodiments, depending on the context, the solid phase cancomprise the well of an assay plate; in others it is a purificationcolumn (e.g., an affinity chromatography column). This term alsoincludes a discontinuous solid phase of discrete particles, such asthose described in U.S. Pat. No. 4,275,149.

[0249] A “liposome” is a small vesicle composed of various types oflipids, phospholipids and/or surfactant which is useful for delivery ofa drug (such as a PRO polypeptide or antibody thereto) to a mammal. Thecomponents of the liposome are commonly arranged in a bilayer formation,similar to the lipid arrangement of biological membranes.

[0250] A “small molecule” is defined herein to have a molecular weightbelow about 500 Daltons TABLE 1 /*  *  * C-C increased from 12 to 15  *Z is average of EQ  * B is average of ND  * match with stop is _M;stop-stop = 0; J (joker) match = 0  */ #define _M −8 /* value of a matchwith a stop */ int _day[26][26] = { /*  A B C D E F G H I J K L M N O PQ R S T U V W X Y Z */ /* A */ {2, 0, −2, 0, 0, −4, 1, −1, −1, 0, −1,−2, −1, 0, _M, 1, 0, −2, 1, 1, 0, 0, −6, 0, −3, 0}, /* B */ {0, 3, −4,3, 2, −5, 0, 1, −2, 0, 0, −3, −2, 2, _M, −1, 1, 0, 0, 0, 0, −2, −5, 0,−3, 1}, /* C */ {−2, −4, 15, −5, −5, −4, −3, −3, −2, 0, −5, −6, −5, −4,_M, −3, −5, −4, 0, −2, 0, −2, −8, 0, 0, −5}, /* D */ {0, 3, −5, 4, 3,−6, 1, 1, −2, 0, 0, −4, −3, 2, _M, −1, 2, −1, 0, 0, 0, −2, −7, 0, −4,2}, /* E */ {0, 2, −5, 3, 4, −5, 0, 1, −2, 0, 0, −3, −2, 1, _M, −1, 2,−1, 0, 0, 0, −2, −7, 0, −4, 3}, /* F */ {−4, −5, −4, −6, −5, 9, −5, −2,1, 0, −5, 2, 0, −4, _M, −5, −5, −4, −3, −3, 0, −1, 0, 0, 7, −5}, /* G */{1, 0, −3, 1, 0, −5, 5, −2, −3, 0, −2, −4, −3, 0, _M, −1, −1, −3, 1, 0,0, −1, −7, 0, −5, 0}, /* H */ {−1, 1, −3, 1, 1, −2, −2, 6, −2, 0, 0, −2,−2, 2, _M, 0, 3, 2, −1, −1, 0, −2, −3, 0, 0, 2}, /* I */ {−1, −2, −2,−2, −2, 1, −3, −2, 5, 0, −2, 2, 2, −2, _M, −2, −2, −2, −1, 0, 0, 4, −5,0, −1, −2}, /* J */ {0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0}, /* K */ {−1, 0, −5, 0, 0, −5, −2, 0, −2, 0,5, −3, 0, 1, _M, −1, 1, 3, 0, 0, 0, −2, −3, 0, −4, 0}, /* L */ {−2, −3,−6, −4, −3, 2, −4, −2, 2, 0, −3, 6, 4, −3, _M, −3, −2, −3, −3 , −1, 0,2, −2, 0, −1, −2} /* M */ {−1, −2, −5, −3, −2, 0, −3, −2, 2, 0, 0, 4, 6,−2, _M, −2, −1, 0, −2, −1, 0, 2, −4, 0, −2, −1}, /* N */ {0, 2, −4, 2,1, −4, 0, 2, −2, 0, 1, −3, −2, 2, _M, −1, 1, 0, 1, 0, 0, −2, −4, 0, −2,1}, /* O */ {_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,0,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,_M,}, /* P */ {1, −1, −3, −1, −1, −5,−1, 0, −2, 0, −1, −3, −2, −1,_M, 6, 0, 0, 1, 0, 0, −1, −6, 0, −5, 0}, /*Q */ {0, 1, −5, 2, 2, −5, −1, 3, −2, 0, 1, −2, −1, 1, _M, 0, 4, 1, −1,−1, 0, −2, −5, 0, −4, 3}, /* R */ {−2, 0, −4, −1, −1, −4, −3, 2, −2, 0,3, −3, 0, 0, _M, 0, 1, 6, 0, −1, 0, −2, 2, 0, −4, 0}, /* S */ {1, 0, 0,0, 0, −3, 1, −1, −1, 0, 0, −3, −2, 1, _M, 1, −1, 0, 2, 1, 0, −1, −2, 0,−3, 0}, /* T */ {1, 0, −2, 0, 0, −3, 0, −1, 0, 0, 0, −1, −1, 0, _M, 0,−1, −1, 1, 3, 0, 0, −5, 0, −3, 0}, /* U */ {0, 0, 0, 0, 0, 0, 0, 0, 0,0, 0, 0, 0, 0,_M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* V */ {0, −2, −2,−2, −2, −1, −1, −2, 4, 0, −2, 2, 2, −2,_M, −1, −2, −2, −1, 0, 0, 4, −6,0, −2, −2}, /* W */ {−6, −5, −8, −7, −7, 0, −7, −3, −5, 0, −3, −2, −4,−4,_M, −6, −5, 2, −2, −5, 0, −6, 17, 0, 0, −6}, /* X */ {0, 0, 0, 0, 0,0, 0, 0, 0, 0, 0, 0, 0, 0, _M, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0, 0}, /* Y */{−3, −3, 0, −4, −4, 7, −5, 0, −1, 0, −4, −1, −2, −2, _M, −5, −4, −4, −3,−3, 0, −2, 0, 0, 10, −4}, /* Z */ {0, 1, −5, 2, 3, −5, 0, 2, −2, 0, 0,−2, −1, 1,_M, 0, 3, 0, 0, 0, 0, −2, −6, 0, −4, 4}, }; /*  */ #include<stdio.h> #include <ctype.h> #define MAXJMP  16 /* max jumps in a diag*/ #define MAXGAP  24 /* don't continue to penalize gaps larger thanthis */ #define JMPS 1024 /* max jmps in an path */ #define MX   4 /*save if there's at least MX-1 bases since last jmp */ #define DMAT   3/* value of matching bases */ #define DMIS   0 /* penalty for mismatchedbases */ #define DINS0   8 /* penalty for a gap */ #define DINS1   1 /*penalty per base */ #define PINS0   8 /* penalty for a gap */ #definePINS1   4 /* penalty per residue */ struct jmp { short n[MAXJMP]; /*size of jmp (neg for dely) */ unsigned short x[MAXJMP]; /* base no. ofjmp in seq x */ /* limits seq to 2{circumflex over ( )}16 −1 */ };struct diag { int score; /* score at last jmp */ long offset; /* offsetof prev block */ short ijmp; /* current jmp index */ struct jmp jp; /*list of jmps */ }; struct path { int spc; /* number of leading spaces */short n[JMPS]; /* size of jmp (gap) */ int x[JMPS]; /* loc of jmp (lastelem before gap) */ }; char *ofile; /* output file name */ char*namex[2]; /* seq names: getseqs() */ char *prog; /* prog name for errmsgs */ char *seqx[2];   /* seqs: getseqs() */ int dmax; /* best diag:nw() */ int dmax0; /* final diag */ int dna; /* set if dna: main() */int endgaps; /* set if penalizing end gaps */ int gapx, gapy; /* totalgaps in seqs */ int len0, len1; /* seq lens */ int ngapx, ngapy; /*total size of gaps */ int smax; /* max score: nw() */ int *xbm; /*bitmap for matching */ long offset; /* current offset in jmp file */struct diag *dx; /* holds diagonals */ struct path pp[2]; /* holds pathfor seqs */ char *calloc(), *malloc(), *index(), *strcpy(); char*getseq(), *g_calloc(); /* Needleman-Wunsch alignment program  *  *usage: progs file1 file2  * where file1 and file2 are two dna or twoprotein sequences.  * The sequences can be in upper- or lower-case anmay contain ambiguity  * Any lines beginning with ‘;’, ‘>’ or ‘<’ areignored  * Max file length is 65535 (limited by unsigned short x in thejmp struct)  * A sequence with ⅓ or more of its elements ACGTU isassumed to be DNA  * Output is in the file “align.out”  *  * The programmay create a tmp file in /tmp to hold info about traceback.  * Originalversion developed under BSD 4.3 on a vax 8650  */ #include “nw.h”#include “day.h” static _dbval[26] = {1,14,2,13,0,0,4,11,0,0,12,0,3,15,0,0,0,5,6,8,8,7,9,0,10,0 }; static_pbval[26] = { 1, 2|(1< <(‘D’-‘A’))|(1< <(‘N’-‘A’)), 4, 8, 16, 32, 64,128, 256, 0×FFFFFFF, 1< <10, 1< <11, 1< <12, 1< <13, 1< <14, 1< <15, 1<<16, 1< <17, 1< <18, 1< <19, 1< <20, 1< <21, 1< <22, 1< <23, 1< <24, 1<<25|(1< <(‘E’-‘A’))|(1< <(‘Q’-‘A’)) }; main(ac, av) main int ac; char*av[]; { prog = av[0]; if(ac != 3) { fprintf(stderr, “usage: %s file1file2\n”, prog); fprintf(stderr, “where file1 and file2 are two dna ortwo protein sequences.\n”); fprintf(stderr, “The sequences can be inupper- or lower-case\n”); fprintf(stderr, “Any lines beginning with ‘;’or ‘<’ are ignored\n”); fprintf(stderr, “Output is in the file\“align.out\”\n”); exit(1); } namex[0] = av[1]; namex[1] = av[2];seqx[0] = getseq(namex[0], &len0); seqx[1] = getseq(namex[1], &len1);xbm = (dna)? _dbval : _pbval; endgaps = 0; /* 1 to penalize endgaps */ofile = “align.out”; /* output file */ nw(); /* fill in the matrix, getthe possible jmps */ readjmps(); /* get the actual jmps */ print(); /*print stats, alignment */ cleanup(0); /* unlink any tmp files */ } /* dothe alignment, return best score: main()  * dna: values in Fitch andSmith, PNAS, 80, 1382-1386, 1983  * pro: PAM 250 values  * When scoresare equal, we prefer mismatches to any gap, prefer  * a new gap toextending an ongoing gap, and prefer a gap in seqx  * to a gap in seq y. */ nw() nw { char *px, *py;   /* seqs and ptrs */ int *ndely, *dely; /*keep track of dely */ int ndelx, delx; /* keep track of delx */ int*tmp; /* for swapping row0, row1 */ int mis; /* score for each type */int ins0, ins1; /* insertion penalties */ register id; /* diagonal index*/ register ij; /* jmp index */ register *col0, *col1; /* score forcurr, last row */ register xx, yy; /* index into seqs */ dx = (structdiag *)g_calloc(“to get diags”, len0+len1+1, sizeof(struct diag)); ndely= (int *)g_calloc(“to get ndely”, len1+1, sizeof(int)); dely = (int*)g_calloc(“to get dely”, len1+1, sizeof(int)); col0 = (int*)g_calloc(“to get col0”, len1+1, sizeof(int)); col1 = (int*)g_calloc(“to get col1”, len1+1, sizeof(int)); ins0 = (dna)? DINS0 :PINS0; ins1 = (dna)? DINS1 : PlNS1; smax = −10000; if (endgaps) { for(col0[0] = dely[0] = −ins0, yy = 1; yy <= len1; yy++) { col0[yy] =dely[yy] = col0[yy−1] − ins1; ndely[yy] = yy; } col0[0] = 0; /* WatermanBull Math Biol 84 */ } else for (yy= 1; yy <= len1; yy++) dely[yy] =−ins0; /* fill in match matrix  */ for (px = seqx[0], xx = 1; xx <=len0; px++, xx++) { /* initialize first entry in col  */ if (endgaps) {if (xx == 1) col1[0] = delx = −(ins0+ins1); else col1[0] = delx =col0[0]−ins1; ndelx = xx; } else { col1[0] = 0; delx = −ins0; ndelx = 0;} ...nw for (py = seqx[1], yy = 1; yy <= len1; py++, yy++) { mis =col0[yy−1]; if (dna) mis + = (xbm[*px−‘A’]&xbm[*py−‘A’])? DMAT : DMIS;else mis += _day[*px−‘A’][*py−‘A’]; /* update penalty for del in x seq; * favor new del over ongong del  * ignore MAXGAP if weighting endgaps */ if (endgaps || ndely[yy] < MAXGAP) { if (col0[yy] − ins0 >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } else {dely[yy] −= ins1; ndely[yy]++; } } else { if (col0[yy] − (ins0+ins1) >=dely[yy]) { dely[yy] = col0[yy] − (ins0+ins1); ndely[yy] = 1; } elsendely[yy]++; } /* update penalty for del in y seq;  * favor new del overongong del  */ if (endgaps || ndelx < MAXGAP) { if(col1[yy−1] − ins0 >=delx) { delx = col1[yy−1] − (ins0+ins1); ndelx = 1; } else { delx −=ins1; ndelx++; } } else { if (col1[yy−1] − (ins0+ins1) >= delx) { delx =col1[yy−1] − (ins0+ins1); ndelx = 1; } else ndelx++; } /* pick themaximum score; we're favoring  * mis over any del and delx over dely  */...nw id = xx − yy + len1 − 1; if (mis >= delx && mis >= dely[yy])col1[yy] = mis; else if (delx >= dely[yy]) { col1[yy] = delx; ij =dx[id].ijmp; if (dx[id].jp.n[0] && (!dna || (ndelx >= MAXJMP && xx >dx[id].jp.x[ij]+MX) || mis > dx[id].score+DINS0)) { dx[id].ijmp++; if(++ij >= MAXJMP) { writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset =offset; offset += sizeof(struct jmp) + sizeof(offset); } }dx[id].jp.n[ij] = ndelx; dx[id].jp.x[ij] = xx; dx[id].score = delx; }else { col1[yy] = dely[yy]; ij = dx[id].ijmp; if (dx[id].jp.n[0] &&(!dna || (ndely[yy] >= MAXJMP && xx > dx[id].jp.x[ij]+MX) || mis >dx[id].score+DINS0)) { dx[id].ijmp++; if (++ij >= MAXJMP) {writejmps(id); ij = dx[id].ijmp = 0; dx[id].offset = offset; offset +=sizeof(struct jmp) + sizeof(offset); } } dx[id].jp.n[ij] =−ndely[yy];dx[id].jp.x[ij] = xx; dx[id].score = dely[yy]; } if (xx == len0 && yy <len1) { /* last col  */ if (endgaps) col1[yy] −= ins0+ins1*(len1−yy);if(col1[yy] > smax) { smax = col1[yy]; dmax = id; } } } if (endgaps &&xx < len0) col1[yy−1] −= ins0+ins1*(len0−xx); if (col1[yy−1] > smax) {smax = col1[yy−1]; dmax = id; } tmp = col0; col0 = col1; col1 = tmp; }(void) free((char *)ndely); (void) free((char *)dely); (void) free((char*)col0); (void) free((char *)col1); } /*  *  * print() -- only routinevisible outside this module  *  * static:  * getmat() -- trace back bestpath, count matches: print()  * pr_align() -- print alignment ofdescribed in array p[]: print()  * dumpblock() -- dump a block of lineswith numbers, stars: pr_align()  * nums() -- put out a number line:dumpblock()  * putline() -- put out a line (name, [num], seq, [num]):dumpblock()  * stars() - -put a line of stars: dumpblock()  *stripname() -- strip any path and prefix from a seqname  */ #include“nw.h” #define SPC  3 #define P_LINE 256 /* maximum output line */#define P_SPC  3 /* space between name or num and seq */ extern_day[26][26]; int olen; /* set output line length */ FILE *fx; /* outputfile */ print() print { int lx, ly, firstgap, lastgap;  /* overlap */ if((fx = fopen(ofile, “w”)) == 0) { fprintf(stderr, “%s: can't write%s\n”, prog, ofile); cleanup(1); } fprintf(fx, “<first sequence: %s(length = %d)\n”, namex[0], len0); fprintf(fx, “<second sequence: %s(length = %d)\n”, namex[1], len1); olen = 60; lx = len0; ly = len1;firstgap = lastgap = 0; if (dmax < len1 − 1) { /* leading gap in x */pp[0].spc = firstgap = len1 − dmax − 1; ly −= pp[0].spc; } else if(dmax > len1 − 1) { /* leading gap in y */ pp[1].spc = firstgap = dmax −(len1 − 1); lx −= pp[1].spc; } if (dmax0 < len0 − 1) { /* trailing gapin x */ lastgap = len0 − dmax0 −1; lx −= lastgap; } else if (dmax0 >len0 − 1) { /* trailing gap in y */ lastgap = dmax0 − (len0 − 1); ly −=lastgap; } getmat(lx, ly, firstgap, lastgap); pr_align(); } /*  * traceback the best path, count matches  */ static getmat(lx, ly, firstgap,lastgap) getmat int lx, ly; /* “core” (minus endgaps) */ int firstgap,lastgap; /* leading trailing overlap */ { int nm, i0, i1, siz0, siz1;char outx[32]; double pct; register n0, n1; register char *p0, *p1; /*get total matches, score  */ i0 = i1 = siz0 = siz1 = 0; p0 = seqx[0] +pp[1].spc; p1 = seqx[1] + pp[0].spc; n0 = pp[1].spc + 1; n1 =pp[0].spc + 1; nm = 0; while ( *p0 && *p1 ) { if (siz0) { p1++; n1++;siz0−−; } else if (siz1) { p0++; n0++; siz1−−; } else { if(xbm[*p0−‘A’]&xbm[*p1−‘A’]) nm++; if (n0++ == pp[0].x[i0]) siz0 =pp[0].n[i0++]; if (nl++ == pp[1].x[i1]) siz1 = pp[1].n[il++]; p0++;p1++; } } /* pct homology:  * if penalizing endgaps, base is the shorterseq  * else, knock off overhangs and take shorter core  */ if (endgaps)lx = (len0 < len1)? len0 : len1; else lx = (lx < ly)? lx : ly; pct =100.*(double)nm/(double)lx; fprintf(fx, “\n”); fprintf(fx, “<%d match%sin an overlap of %d: %.2f percent similarity\n”, nm, (nm == 1)? “” :“es”, lx, pct); fprintf(fx, “<gaps in first sequence: %d”, gapx);...getmat if (gapx) { (void) sprintf(outx, “(%d %s%s)”, ngapx, (dna)?“base”: “residue”, (ngapx == 1)? “”:“s”); fprintf(fx, “% s”, outx);fprintf(fx, “, gaps in second sequence: %d”, gapy); if (gapy) { (void)sprintf(outx, “(%d %s%s)”, ngapy, (dna)? “base”:“residue”, (ngapy == 1)?“”:“s”); fprintf(fx, “%s”, outx); } if (dna) fprintf(fx, “\n<score: %d(match = %d, mismatch = %d, gap penalty = %d + %d per base)\n”, smax,DMAT, DMIS, DINS0, DINS1); else fprintf(fx, “\n<score: %d (Dayhoff PAM250 matrix, gap penalty = %d + %d per residue)\n”, smax, PINS0, PINS1);if (endgaps) fprintf(fx, “<endgaps penalized. left endgap: %d %s%s,right endgap: %d %s%s\n”, firstgap, (dna)? “base” : “residue”, (firstgap== 1)? “” : “s”, lastgap, (dna)? “base” : “residue”, (lastgap == 1)? “”: “s”); else fprintf(fx, “<endgaps not penalized\n”); } static nm; /*matches in core -- for checking */ static lmax; /* lengths of strippedfile names */ static ij[2]; /* jmp index for a path */ static nc[2]; /*number at start of current line */ static ni[2]; /* current elem number-- for gapping */ static siz[2]; static char *ps[2]; /* ptr to currentelement */ static char *po[2]; /* ptr to next output char slot */ staticchar out[2][P_LINE]; /* output line */ static char star[P_LINE]; /* setby stars() */ /*  * print alignment of described in struct path pp[]  */static pr_align() pr_align { int nn; /* char count */ int more; registeri; for (i = 0, lmax = 0; i < 2++) { nn = stripname(namex[i]); if (nn >lmax) lmax = nn; nc[i] = 1; ni[i] = 1; siz[i] = ij[i] = 0; ps[i] =seqx[i]; po[i] = out[i]; } for (nn = nm = 0, more = 1; more;) {...pr_align for (i = more = 0; i < 2; i++) { /*  * do we have more ofthis sequence?  */ if (!*ps[i]) continue; more++; if (pp[i].spc) { /*leading space */ *po[i]++ = ‘ ’; pp[i].spc−−; } else if (siz[i]) { /* ina gap */ *po[i]++ = ‘−’; siz[i]−−; } else { /* we're putting a seqelement */ *po[i] = *ps[i]; if (islower(*ps[i])) *ps[i] =toupper(*ps[i]); po[i]++; ps[i]++; /*  * are we at next gap for thisseq?  */ if (ni[i] == pp[i].x[ij[i]]) { /*  * we need to merge all gaps * at this location  */ siz[i] == pp[i].n[ij[i]++]; while (ni[i] ==pp[i].x[ij[i]]) siz[i] += pp[i].n[ij[i]++]; } ni[i]++; } } if (++nn ==olen || !more && nn) { dumpblock(); for (i = 0; i < 2; i++) po[i] =out[i]; nn = 0; } } } /*  * dump a block of lines, including numbers,stars: pr_align()  */ static dumpblock() dumpblock { register i; for(i =0; i < 2; i++) *po[i]−− = ‘\0’; ...dumpblock (void) putc(‘\n’, fx); for(i = 0; i < 2; i++) { if (*out[i] && (*out[i] != ‘ ’ || *(po[i]) != ‘’)) { if (i == 0) nums(i); if (i == 0 && *out[1]) stars(); putline(i);if (i == 0 && *out[1]) fprintf(fx, star); if (i == 1) nums(i); } } } *put out a number line: dumpblock()  */ static nums(ix) nums int  ix; /*index in out[] holding seq line */ { char nline[P_LINE]; register i, j;register char *pn, *px, *py; for(pn = nline, i = 0; i < lmax+P_SPC; i++,pn++) *pn = ‘ ’; for (i = nc[ix], py = out[ix]; *py; py++, pn++) { if(*py == ‘ ’ || *py == ‘−’); *pn = ‘ ’; else { if (i%10 == 0 || (i == 1&& nc[ix] != 1)) { j = (i < 0)? −i : i; for (px = pn; j; j/= 10, px−−)*px = j%10 + ‘0’; if (i < 0) *px = ‘−’; } else *pn = ‘ ’; i++; } } *pn =‘\0’; nc[ix] = i; for (pn = nline; *pn; pn++) (void) putc(*pn, fx);(void) putc(‘\n’, fx); } /*  * put out a line (name, [num], seq. [num]):dumpblock()  */ static putline(ix) putline int   ix; { ...putline int i;register char *px; for (px = namex[ix], i = 0; *px && *px != ‘:’; px++,i++) (void) putc(*px, fx); for (;i < lmax+P_SPC; i++) (void) putc(‘ ’,fx); /* these count from 1:  * ni[] is current element (from 1)  * nc[]is number at start of current line  */ for (px = out[ix]; *px; px++)(void) putc(*px&0x7F, fx); (void) putc(‘\n’, fx); } /*  * put a line ofstars (seqs always in out[0], out[1]): dumpblock()  */ static stars()stars { int i; register char *p0, *p1, cx, *px; if (!*out[0] || (*out[0]== ‘ ’ && *(p0[0]) == ‘ ’) || !*out[1] || (*out[1] == ‘ ’ && *(po[1]) ==‘ ’)) return; px = star; for (i = lmax+P_SPC; i; i−−) *px++ = ‘ ’; for(p0 = out[0], p1 = out[1]; *p0 && *p1; p0++, p1++) { if (isalpha(*p0) &&isalpha(*p1)) { if (xbm[*p0−‘A’]&xbm[*p1−‘A’]) { cx = ‘*’; nm++; } elseif (!dna && _day[*p0− ‘A’][*p1−‘A’] > 0) cx = ‘.’; else cx = ‘ ’; } elsecx = ‘ ’; *px++ = cx; } *px++ = ‘\n’; *px = ‘\0’; } /*  * strip path orprefix from pn, return len: pr_align()  */ static stripname(pn)stripname char *pn; /* file name (may be path) */ { register char *px,*py; py = 0; for (px = pn; *px; px++) if (*px == ‘/’) py = px + 1; if(py) (void) strcpy(pn, py); return(strlen(pn)); } /*  * cleanup() --cleanup any tmp file  * getseq() -- read in seq, set dna, len, maxlen  *g_calloc() -- calloc() with error checkin  * readjmps() -- get the goodjmps, from tmp file if necessary  * writejmps() -- write a filled arrayof jmps to a tmp file: nw()  */ #include “nw.h” #include <sys/file.h>char *jname = “/tmp/homgXXXXXX”; /* tmp file for jmps */ FILE *fj; intcleanup(); /* cleanup tmp file */ long lseek(); /*  * remove any tmpfile if we blow  */ cleanup(i) cleanup int i; { if (fj) (void)unlink(jname); exit(i); } /*  * read, return ptr to seq, set dna, len,maxlen  * skip lines starting with ‘;’, ‘<’, or ‘>’  * seq in upper orlower case  */ char * getseq(file, len) getseq char *file; /* file name*/ int *len; /* seq len */ { char line[1024], *pseq; register char *px,*py; int natgc, tlen; FILE *fp; if ((fp = fopen(file, “r”)) == 0) {fprintf(stderr, “%s: can't read %s\n”, prog, file); exit(1); } tlen =natgc = 0; while (fgets(line, 1024, fp)) { if (*line == ‘;’ || *line ==‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’; px++) if(isupper(*px) || islower(*px)) tlen++; } if ((pseq =malloc((unsigned)(tlen+6))) == 0) { fprintf(stderr, “%s: malloc() failedto get %d bytes for %s\n”, prog, tlen+6, file); exit(1); } pseq[0] =pseq[1] = pseq[2] = pseq[3] = ‘\0’; ...getseq py = pseq + 4; *len =tlen; rewind(fp); while (fgets(line, 1024, fp)) { if (*line == ‘;’ ||*line == ‘<’ || *line == ‘>’) continue; for (px = line; *px != ‘\n’;px++) { if (isupper(*px)) *py++ = *px; else if (islower(*px)) *py++ =toupper(*px); if (index(“ATGCU”, *(py−1))) natgc++; } } *py++ = ‘\0’;*py = ‘\0’; (void) fclose(fp); dna = natgc > (tlen/3); return(pseq+4); }char * g_calloc(msg, nx, sz) g_calloc char *msg; /* program, callingroutine */ int nx, sz; /* number and size of elements */ { char *px,*calloc(); if ((px = calloc((unsigned)nx, (unsigned)sz)) == 0) { if(*msg) { fprintf(stderr, “%s: g_calloc() failed %s (n= %d, sz= %d)\n”,prog, msg, nx, sz); exit(1); } } return(px); } /*  * get final jmps fromdx[] or tmp file, set pp[], reset dmax: main()  */ readjmps() readjmps {int fd = −1; int siz, i0, i1; register i, j, xx; if (fj) { (void)fclose(fj); if ((fd = open(jname, O_RDONLY, 0)) < 0) { fprintf(stderr,“%s: can't open() %s\n”, prog, jname); cleanup(1); } } for (i = i0 = i1= 0, dmax0 = dmax, xx = len0; ;i++) { while (1) { for (j =dx[dmax].ijmp; j >= 0 && dx[dmax].jp.x[j] >= xx; j−−) ; ...readjmps if(j < 0 && dx[dmax].offset && fj) { (void) lseek(fd, dx[dmax].offset, 0);(void) read(fd, (char *)&dx[dmax].jp, sizeof(struct jmp)); (void)read(fd, (char *)&dx[dmax].offset, sizeof(dx[dmax].offset));dx[dmax].ijmp = MAXJMP−1; } else break; } if (i >= JMPS) {fprintf(stderr, “%s: too many gaps in alignment\n”, prog); cleanup(1); }if (j >= 0) { siz = dx[dmax].jp.n[j]; xx = dx[dmax].jp.x[j]; dmax +=siz; if (siz < 0) { /* gap in second seq */ pp[1].n[il] = −siz; xx +=siz; /* id = xx − yy + len1 − 1  */ pp[1].x[il] = xx − dmax + len1 − 1;gapy++; ngapy −= siz; /* ignore MAXGAP when doing endgaps */ siz = (−siz< MAXGAP || endgaps)? −siz : MAXGAP; il++; } else if (siz > 0) { /* gapin first seq */ pp[0] .n[i0] = siz; pp[0] .x[i0] = xx; gapx++; ngapx +=siz; /* ignore MAXGAP when doing endgaps */ siz = (siz < MAXGAP ||endgaps)? siz : MAXGAP; i0++; } } else break; } /* reverse the order ofjmps  */ for (j = 0, i0−−; j < i0; j++, i0−−) { i = pp[0].n[j];pp[0].n[j] = pp[0].n[i0]; pp[0].n[i0] = i; i = pp[0].x[j]; pp[0].x[j] =pp[0].x[i0]; pp[0].x[i0] = i; } for (j = 0, i1−−; j < i1; j++, i1−−) { i= pp[1].n[j]; pp[1].n[j] = pp[1].n[i1]; pp[1].n[i1] = i; i = pp[1].x[j];pp[1].x[j] = pp[1].x[i1]; pp[1].x[i1] = i; } if (fd >= 0) (void)close(fd); if (fj) { (void) unlink(jname); fj = 0; offset = 0; } } /*  *write a filled jmp struct offset of the prev one (if any): nw()  */writejmps(ix) writejmps int ix; { char *mktemp(); if (!fj) { if(mktemp(jname) < 0) { fprintf(stderr, “%s: can't mktemp() %s\n”, prog,jname); cleanup(1); } if ((fj = fopen(jname, “w”)) == 0) {fprintf(stderr, “%s: can't write %s\n”, prog, jname); exit(1); } }(void) fwrite((char *)&dx[ix].jp, sizeof(struct jmp), 1, fj); (void)fwrite((char *)&dx[ix].offset, sizeof(dx[ix].offset), 1, fj); }

[0251] TABLE 2 PRO XXXXXXXXXXXXXXX (Length = 15 amino acids) ComparisonProtein XXXXXYYYYYYY (Length = 12 amino acids)

[0252] TABLE 3 PRO XXXXXXXXXX (Length = 10 amino acids) ComparisonProtein XXXXXYYYYYYZZYZ (Length = 15 amino acids)

[0253] TABLE 4 PRO-DNA NNNNNNNNNNNNNN (Length = 14 nucleotides)Comparison DNA NNNNNNLLLLLLLLLL (Length = 16 nucleotides)

[0254] TABLE 5 PRO-DNA NNNNNNNNNNNN (Length = 12 nucleotides) ComparisonDNA NNNNLLLVV (Length = 9 nucleotides)

[0255] II. Compositions and Methods of the Invention

[0256] A. Full-Length PRO Polypeptides

[0257] The present invention provides newly identified and isolatednucleotide sequences encoding polypeptides referred to in the presentapplication as PRO polypeptides. In particular, cDNAs encoding variousPRO polypeptides have been identified and isolated, as disclosed infurther detail in the Examples below. It is noted that proteins producedin separate expression rounds may be given different PRO numbers but theUNQ number is unique for any given DNA and the encoded protein, and willnot be changed. However, for sake of simplicity, in the presentspecification the protein encoded by the full length native nucleic acidmolecules disclosed herein as well as all further native homologues andvariants included in the foregoing definition of PRO, will be referredto as “PRO/number”, regardless of their origin or mode of preparation.

[0258] As disclosed in the Examples below, various cDNA clones have beendeposited with the ATCC. The actual nucleotide sequences of those clonescan readily be determined by the skilled artisan by sequencing of thedeposited clone using routine methods in the art. The predicted aminoacid sequence can be determined from the nucleotide sequence usingroutine skill. For the PRO polypeptides and encoding nucleic acidsdescribed herein, Applicants have identified what is believed to be thereading frame best identifiable with the sequence information availableat the time.

[0259] B. PRO Polypeptide Variants

[0260] In addition to the full-length native sequence PRO polypeptidesdescribed herein, it is contemplated that PRO variants can be prepared.PRO variants can be prepared by introducing appropriate nucleotidechanges into the PRO DNA, and/or by synthesis of the desired PROpolypeptide. Those skilled in the art will appreciate that amino acidchanges may alter post-translational processes of the PRO, such aschanging the number or position of glycosylation sites or altering themembrane anchoring characteristics.

[0261] Variations in the native full-length sequence PRO or in variousdomains of the PRO described herein, can be made, for example, using anyof the techniques and guidelines for conservative and non-conservativemutations set forth, for instance, in U.S. Pat. No. 5,364,934.Variations may be a substitution, deletion or insertion of one or morecodons encoding the PRO that results in a change in the amino acidsequence of the PRO as compared with the native sequence PRO. Optionallythe variation is by substitution of at least one amino acid with anyother amino acid in one or more of the domains of the PRO. Guidance indetermining which amino acid residue may be inserted, substituted ordeleted without adversely affecting the desired activity may be found bycomparing the sequence of the PRO with that of homologous known proteinmolecules and minimizing the number of amino acid sequence changes madein regions of high homology. Amino acid substitutions can be the resultof replacing one amino acid with another amino acid having similarstructural and/or chemical properties, such as the replacement of aleucine with a serine, i.e., conservative amino acid replacements.Insertions or deletions may optionally be in the range of about 1 to 5amino acids. The variation allowed may be determined by systematicallymaking insertions, deletions or substitutions of amino acids in thesequence and testing the resulting variants for activity exhibited bythe full-length or mature native sequence.

[0262] PRO polypeptide fragments are provided herein. Such fragments maybe truncated at the N-terminus or C-terminus, or may lack internalresidues, for example, when compared with a full length native protein.Certain fragments lack amino acid residues that are not essential for adesired biological activity of the PRO polypeptide.

[0263] PRO fragments may be prepared by any of a number of conventionaltechniques. Desired peptide fragments may be chemically synthesized. Analternative approach involves generating PRO fragments by enzymaticdigestion, e.g., by treating the protein with an enzyme known to cleaveproteins at sites defined by particular amino acid residues, or bydigesting the DNA with suitable restriction enzymes and isolating thedesired fragment. Yet another suitable technique involves isolating andamplifying a DNA fragment encoding a desired polypeptide fragment, bypolymerase chain reaction (PCR). Oligonucleotides that define thedesired termini of the DNA fragment are employed at the 5′ and 3′primers in the PCR. Preferably, PRO polypeptide fragments share at leastone biological and/or immunological activity with the native PROpolypeptide disclosed herein.

[0264] In particular embodiments, conservative substitutions of interestare shown in Table 6 under the heading of preferred substitutions. Ifsuch substitutions result in a change in biological activity, then moresubstantial changes, denominated exemplary substitutions in Table 6, oras further described below in reference to amino acid classes, areintroduced and the products screened. TABLE 6 Original ExemplaryPreferred Residue Substitutions Substitutions Ala (A) val; leu; ile valArg (R) lys; gln; asn lys Asn (N) gln; his; lys; arg gln Asp (D) glu gluCys (C) ser ser Gln (Q) asn asn Glu (E) asp asp Gly (G) pro; ala ala His(H) asn; gln; lys; arg arg Ile (I) leu; val; met; ala; phe leunorleucine Leu (L) norleucine, ile; val; ile met, ala, phe Lys (K) arg;gln; asn arg Met (M) leu; phe; ile leu Phe (F) leu; val; ile; ala; tyrleu Pro (P) ala ala Ser (S) thr thr Thr (T) ser ser Trp (W) tyr; phe tyrTyr (Y) trp; phe; thr; ser phe Val (V) ile; leu, met, phe; leu ala,norleucine

[0265] Substantial modifications in function or immunological identityof the PRO polypeptide are accomplished by selecting substitutions thatdiffer significantly in their effect on maintaining (a) the structure ofthe polypeptide backbone in the area of the substitution, for example,as a sheet or helical conformation, (b) the charge or hydrophobicity ofthe molecule at the target site, or (c) the bulk of the side chain.

[0266] Naturally occurring residues are divided into groups based oncommon side-chain properties:

[0267] (1) hydrophobic: norleucine, met, ala, val, leu, ile;

[0268] (2) neutral hydrophilic: cys, ser, thr;

[0269] (3) acidic: asp, glu;

[0270] (4) basic: asn, gin, his, lys, arg;

[0271] (5) residues that influence chain orientation: gly, pro; and

[0272] (6) aromatic: trp, tyr, phe.

[0273] Non-conservative substitutions will entail exchanging a member ofone of these classes for another class. Such substituted residues alsomay be introduced into the conservative substitution sites or, morepreferably, into the remaining (non-conserved) sites.

[0274] The variations can be made using methods known in the art such asoligonucleotide-mediated (site-directed) mutagenesis, alanine scanning,and PCR mutagenesis. Site-directed mutagenesis [Carter et al., Nucl.Acids Res., 13:4331 (1986); Zoller et al., Nucl. Acids Res., 10:6487(1987)], cassette mutagenesis [Wells et al., Gene, 34:315 (1985)],restriction selection mutagenesis [Wells et al., Philos. Trans. R. Soc.London SerA, 317:415 (1986)] or other known techniques can be performedon the cloned DNA to produce the PRO variant DNA.

[0275] Scanning amino acid analysis can also be employed to identify oneor more amino acids along a contiguous sequence. Among the preferredscanning amino acids are relatively small, neutral amino acids. Suchamino acids include alanine, glycine, serine, and cysteine. Alanine istypically a preferred scanning amino acid among this group because iteliminates the side-chain beyond the beta-carbon and is less likely toalter the main-chain conformation of the variant [Cunningham and Wells,Science, 244: 1081-1085 (1989)]. Alanine is also typically preferredbecause it is the most common amino acid. Further, it is frequentlyfound in both buried and exposed positions [Creighton, The Proteins,(W.H. Freeman & Co., N.Y.); Chothia, J. Mol. Biol., 150:1 (1976)]. Ifalanine substitution does not yield adequate amounts of variant, anisoteric amino acid can be used.

[0276] C. Modifications of PRO

[0277] Covalent modifications of PRO are included within the scope ofthis invention. One type of covalent modification includes reactingtargeted amino acid residues of a PRO polypeptide with an organicderivatizing agent that is capable of reacting with selected side chainsor the N- or C-terminal residues of the PRO. Derivatization withbifunctional agents is useful, for instance, for crosslinking PRO to awater-insoluble support matrix or surface for use in the method forpurifying anti-PRO antibodies, and vice-versa. Commonly usedcrosslinking agents include, e.g., 1,1-bis (diazoacetyl)-2-phenylethane,glutaraldehyde, N-hydroxysuccinimide esters, for example, esters with4-azidosalicylic acid, homobifunctional imidoesters, includingdisuccinimidyl esters such as 3,3′-dithiobis(succinimidylpropionate),bifunctional maleimides such as bis-N-maleimido-1,8-octane and agentssuch as methyl-3-[(p-azidophenyl)dithio]propioimidate.

[0278] Other modifications include deamidation of glutaminyl andasparaginyl residues to the corresponding glutamyl and aspartylresidues, respectively, hydroxylation of proline and lysine,phosphorylation of hydroxyl groups of seryl or threonyl residues,methylation of the or α-amino groups of lysine, arginine, and histidineside chains [T.E. Creighton, Proteins: Structure and MolecularProperties, W.H. Freeman & Co., San Francisco, pp. 79-86 (1983)],acetylation of the N-terminal amine, and amidation of any C-terminalcarboxyl group.

[0279] Another type of covalent modification of the PRO polypeptideincluded within the scope of this invention comprises altering thenative glycosylation pattern of the polypeptide. “Altering the nativeglycosylation pattern” is intended for purposes herein to mean deletingone or more carbohydrate moieties found in native sequence PRO (eitherby removing the underlying glycosylation site or by deleting theglycosylation by chemical and/or enzymatic means), and/or adding one ormore glycosylation sites that are not present in the native sequencePRO. In addition, the phrase includes qualitative changes in theglycosylation of the native proteins; involving a change in the natureand proportions of the various carbohydrate moieties present.

[0280] Addition of glycosylation sites to the PRO polypeptide may beaccomplished by altering the amino acid sequence. The alteration may bemade, for example, by the addition of, or substitution by, one or moreserine or threonine residues to the native sequence PRO (for O-linkedglycosylation sites). The PRO amino acid sequence may optionally bealtered through changes at the DNA level, particularly by mutating theDNA encoding the PRO polypeptide at preselected bases such that codonsare generated that will translate into the desired amino acids.

[0281] Another means of increasing the number of carbohydrate moietieson the PRO polypeptide is by chemical or enzymatic coupling ofglycosides to the polypeptide. Such methods are described in the art,e.g., in WO 87/05330 published Sep. 11, 1987, and in Aplin and Wriston,CRC Crit. Rev. Biochem., pp. 259-306 (1981).

[0282] Removal of carbohydrate moieties present on the PRO polypeptidemay be accomplished chemically or enzymatically or by mutationalsubstitution of codons encoding for amino acid residues that serve astargets for glycosylation. Chemical deglycosylation techniques are knownin the art and described, for instance, by Hakimuddin, et al., Arch.Biochem. Biophys., 259:52 (1987) and by Edge et al., Anal. Biochem.,118:131 (1981). Enzymatic cleavage of carbohydrate moieties onpolypeptides can be achieved by the use of a variety of endo- andexo-glycosidases as described by Thotakura et al., Meth. Enzymol.,138:350 (1987).

[0283] Another type of covalent modification of PRO comprises linkingthe PRO polypeptide to one of a variety of nonproteinaceous polymers,e.g., polyethylene glycol (PEG), polypropylene glycol, orpolyoxyalkylenes, in the manner set forth in U.S. Pat. Nos. 4,640,835;4,496,689; 4,301,144; 4,670,417; 4,791,192 or 4,179,337.

[0284] The PRO of the present invention may also be modified in a way toform a chimeric molecule comprising PRO fused to another, heterologouspolypeptide or amino acid sequence.

[0285] In one embodiment, such a chimeric molecule comprises a fusion ofthe PRO with a tag polypeptide which provides an epitope to which ananti-tag antibody can selectively bind. The epitope tag is generallyplaced at the amino- or carboxyl-terminus of the PRO. The presence ofsuch epitope-tagged forms of the PRO can be detected using an antibodyagainst the tag polypeptide. Also, provision of the epitope tag enablesthe PRO to be readily purified by affinity purification using ananti-tag antibody or another type of affinity matrix that binds to theepitope tag. Various tag polypeptides and their respective antibodiesare well known in the art. Examples include poly-histidine (poly-his) orpoly-histidine-glycine (poly-his-gly) tags; the flu HA tag polypeptideand its antibody 12CA5 [Field et al., Mol. Cell. Biol., 8:2159-2165(1988)]; the c-myc tag and the 8F9, 3C7, 6E10, G4, B7 and 9E10antibodies thereto [Evan et al., Molecular and Cellular Biology,5:3610-3616 (1985)]; and the Herpes Simplex virus glycoprotein D (gD)tag and its antibody [Paborsky et al., Protein Engineering, 3(6):547-553(19903]. Other tag polypeptides include the Flag-peptide [Hopp et al.,BioTechnology, 6:1204-1210 (1988)]; the KT3 epitope peptide [Martin etal., Science, 255:192-194 (1992)]; an α-tubulin epitope peptide [Skinneret al., J. Biol. Chemi., 266:15163-15166 (1991)]; and the T7 gene 10protein peptide tag [Lutz-Freyermuth et al., Proc. Natl. Acad. Sci. USA,87:6393-6397 (1990)].

[0286] In an alternative embodiment, the chimeric molecule may comprisea fusion of the PRO with an immunoglobulin or a particular region of animmunoglobulin. For a bivalent form of the chimeric molecule (alsoreferred to as an “immunoadhesin”), such a fusion could be to the Fcregion of an IgG molecule. The 1 g fusions preferably include thesubstitution of a soluble (transmembrane domain deleted or inactivated)form of a PRO polypeptide in place of at least one variable regionwithin an Ig molecule. In a particularly preferred embodiment, theimmunoglobulin fusion includes the hinge, CH2 and CH3, or the hinge,CH1, CH2 and CH3 regions of an IgGI molecule. For the production ofimmunoglobulin fusions see also U.S. Pat. No. 5,428,130 issued Jun. 27,1995.

[0287] D. Preparation of PRO

[0288] The description below relates primarily to production of PRO byculturing cells transformed or transfected with a vector containing PROnucleic acid. It is, of course, contemplated that alternative methods,which are well known in the art, may be employed to prepare PRO. Forinstance, the PRO sequence, or portions thereof, may be produced bydirect peptide synthesis using solid-phase techniques [see, e.g.,Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., SanFrancisco, Calif. (1969); Merrifield, J. Am. Chem. Soc., 85:2149-2154(1963)]. In vitro protein synthesis may be performed using manualtechniques or by automation. Automated synthesis may be accomplished,for instance, using an Applied Biosystems Peptide Synthesizer (FosterCity, Calif.) using manufacturer's instructions. Various portions of thePRO may be chemically synthesized separately and combined using chemicalor enzymatic methods to produce the full-length PRO.

[0289] 1. Isolation of DNA Encoding PRO

[0290] DNA encoding PRO may be obtained from a cDNA library preparedfrom tissue believed to possess the PRO mRNA and to express it at adetectable level. Accordingly, human PRO DNA can be convenientlyobtained from a cDNA library prepared from human tissue, such asdescribed in the Examples. The PRO-encoding gene may also be obtainedfrom a genomic library or by known synthetic procedures (e.g., automatednucleic acid synthesis).

[0291] Libraries can be screened with probes (such as antibodies to thePRO or oligonucleotides of at least about 20-80 bases) designed toidentify the gene of interest or the protein encoded by it. Screeningthe cDNA or genomic library with the selected probe may be conductedusing standard procedures, such as described in Sambrook. et al.,Molecular Cloning: A Laboratory Manual (New York: Cold Spring HarborLaboratory Press, 1989). An alternative means to isolate the geneencoding PRO is to use PCR methodology [Sambrook et al., supra;Dieffenbach et al., PCR Primer: A Laboratory Manual (Cold Spring HarborLaboratory Press, 1995)].

[0292] The Examples below describe techniques for screening a cDNAlibrary. The oligonucleotide sequences selected as probes should be ofsufficient length and sufficiently unambiguous that false positives areminimized. The oligonucleotide is preferably labeled such that it can bedetected upon hybridization to DNA in the library being screened.Methods of labeling are well known in the art, and include the use ofradiolabels like ³²P-labeled ATP, biotinylation or enzyme labeling.Hybridization conditions, including moderate stringency and highstringency, are provided in Sambrook et al., supra.

[0293] Sequences identified in such library screening methods can becompared and aligned to other known sequences deposited and available inpublic databases such as GenBank or other private sequence databases.Sequence identity (at either the amino acid or nucleotide level) withindefined regions of the molecule or across the full-length sequence canbe determined using methods known in the art and as described herein.

[0294] Nucleic acid having protein coding sequence may be obtained byscreening selected cDNA or genomic libraries using the deduced aminoacid sequence disclosed herein for the first time, and, if necessary,using conventional primer extension procedures as described in Sambrooket al., supra, to detect precursors and processing intermediates of mRNAthat may not have been reverse-transcribed into cDNA.

[0295] 2. Selection and Transformation of Host Cells

[0296] Host cells are transfected or transformed with expression orcloning vectors described herein for PRO production and cultured inconventional nutrient media modified as appropriate for inducingpromoters, selecting transformants, or amplifying the genes encoding thedesired sequences. The culture conditions, such as media, temperature,pH and the like, can be selected by the skilled artisan without undueexperimentation. In general, principles, protocols, and practicaltechniques for maximizing the productivity of cell cultures can be foundin Mammalian Cell Biotechnology: a Practical Approach, M. Butler, ed.(IRL Press, 1991) and Sambrooket al., supra.

[0297] Methods of eukaryotic cell transfection and prokaryotic celltransformation are known to the ordinarily skilled artisan, for example,CaCl₂, CaPO₄, liposome-mediated and electroporation. Depending on thehost cell used, transformation is performed using standard techniquesappropriate to such cells. The calcium treatment employing calciumchloride, as described in Sambrook et al., supra, or electroporation isgenerally used for prokaryotes. Infection with Agrobacterium tumefaciensis used for transformation of certain plant cells, as described by Shawet al., Gene, 23:315 (1983) and WO 89/05859 published Jun. 29, 1989. Formammalian cells without such cell walls, the calcium phosphateprecipitation method of Graham and van der Eb, Virology, 52:456-457(1978) can be employed. General aspects of mammalian cell host systemtransfections have been described in U.S. Pat. No. 4,399,216.Transformations into yeast are typically carried out according to themethod of Van Solingen et al., J. Bact., 130:946 (1977) and Hsiao etal., Proc. Natl. Acad. Sci. (USA), 76:3829 (1979). However, othermethods for introducing DNA into cells, such as by nuclearmicroinjection, electroporation, bacterial protoplast fusion with intactcells, or polycations, e.g., polybrene, polyornithine, may also be used.For various techniques for transforming mammalian cells, see Keown etal., Methods in Enzymology, 185:527-537 (1990) and Mansour et al.,Nature, 336:348-352 (1988).

[0298] Suitable host cells for cloning or expressing the DNA in thevectors herein include prokaryote, yeast, or higher eukaryote cells.Suitable prokaryotes include but are not limited to eubacteria, such asGram-negative or Gram-positive organisms, for example,Enterobacteriaceae such as E. coli. Various E.coli strains are publiclyavailable, such as E. coli K12 strain MM294 (ATCC 31,446); E coli X1776(ATCC 31,537); E: constrain W3110 (ATCC 27,325) and K5772 (ATCC 53,635).Other suitable prokaryotic host cells include Enterobacteriaceae such asEscherichia, e.g., E. coli, Enteiobacter, Erwinia, Klebsiella, Proteus,Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratiamarcescans, and Shigella, as well as Bacilli such as B. subtilis and B.licheniformis (e.g., B. licheniformis 41P disclosed in DD 266,710published Apr. 12, 1989), Pseudomonas such as P. aeruginosa, andStreptomyces. These examples are illustrative rather than limiting.Strain W3110 is one particularly preferred host or parent host becauseit is a common host strain for recombinant DNA product fermentations.Preferably, the host cell secretes minimal amounts of proteolyticenzymes. For example, strain W3110 may be modified to effect a geneticmutation in the genes encoding proteins endogenous to the host, withexamples of such hosts including E. coli W3110 strain 1A2, which has thecomplete genotype tonA; E. coli W310 strain 9E4, which has the completegenotype tonA ptr3; E. coli W3110 strain 27C7 (ATCC 55,244), which hasthe complete genotype tonA ptr3 phoA E15(argF-lac)769 degP ompT kan^(r); E. coliW3110 strain 37D6, which has the complete genotype tonA ptr3phoA E75 (argF-lac)769 degP ompT rbs7 ilvG kan^(r) ; E. coliW3110 strain40B4, which is strain 37D6 with a non-kanamycin resistant degP deletionmutation; and an E constrain having mutant periplasmic proteasedisclosed in U.S. Pat. No. 4,946,783 issued Aug. 7, 1990. Alternatively,in vitro methods of cloning, e.(g., PCR or other nucleic acid polymerasereactions, are suitable.

[0299] In addition to prokaryotes, eukaryotic microbes such asfilamentous fungi or yeast are suitable cloning or expression hosts forPRO-encoding vectors. Saccharomyces cerevisiae is a commonly used lowereukaryotic host microorganism. Others include Schizosaccharomyces pombe(Beach and Nurse, Nature, 290: 140 [19811; EP 139,383 published May 2,1985); Kluyveromyces hosts (U.S. Pat. No. 4,943,529; Fleer et al.,Bio/Technology, 9:968-975 (1991)) such as, e.g., K. lactis (MW98-8C,CBS683, CBS4574; Louvencourt et al., J. Bacteriol.,154(2):737-742[1983]), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K.drosophilarum (ATCC 36,906; Van den Berg et al., Bio/Technology, 8:135(1990)), K. thermotolerans, and K. marxianus; yarrowia (EP 402,226);Pichia pastoris (EP 183,070; Sreekrishna et al., J. Basic Microbiol.,28:265-278[1988]); Candida; Trichoderina reesia (EP 244,234); Neurosporacrassa (Case et al., Proc. Natl. Acad. Sci. USA, 76:5,259-5263[1979]);Schwanniomyces such as Schwanniomyces occidentalis (EP 394,538 publishedOct. 31, 1990); and filamentous fungi such as, e.g., Neurospora,Penicilium, Tolypocladium (WO 91/00357 published Jan. 1, 1991), andAspeirgillus hosts such as A. nidulans (Ballance et al., Biochem.Biophys. Res. Commun., 112:284-289[1983]; Tilburn et al., Gene,26:205-221[1983]; Yelton et al., Proc. Natl. Acad. Sci. USA, 81:1470-1474[1984]) and A. niger (Kelly and Hynes, EMBO J.,4:475-479[1985]). Methylotropic yeasts are suitable herein and include,but are not limited to, yeast capable of growth on methanol selectedfrom the genera consisting of Hansenula, Candida, Kloeckera, Pichia,Saccharomyces, Torulopsis, and Rhodotoruila. A list of specific speciesthat are exemplary of this class of yeasts may be found in C. Anthony,The Biochemistry of Methylotrophs, 269 (1982).

[0300] Suitable host cells for the expression of glycosylated PRO arederived from multicellular organisms. Examples of invertebrate cellsinclude insect cells such as Drosophila S2 and Spodoptera Sf9, as wellas plant cells. Examples of useful mammalian host cell lines includeChinese hamster ovary (CHO) and COS cells. More specific examplesinclude monkey kidney CV1 line transformed by SV40 (COS-7, ATCC CRL1651); human embryonic kidney line (293 or 293 cells subcloned forgrowth in suspension culture, Graham et al., J. Gen Virol., 36:59(1977)); Chinese hamster ovary cells/-DHFR (CHO, Urlaub and Chasin,Proc. Natl. Acad. Sci. USA, 77:4216 (1980)); mouse sertoli cells (TM4,Mather, Biol. Reprod., 23:243-251 (1980)); human lung cells (Wi 38, ATCCCCL 75); human liver cells (Hep G2, HB 8065); and mouse mammary tumor(MMT 060562, ATCC CCL51). The selection of the appropriate host cell isdeemed to be within the skill in the art.

[0301] 3. Selection and Use of a Replicable Vector

[0302] The nucleic acid (e.g., cDNA or genomic DNA) encoding PRO may beinserted into a replicable vector for cloning (amplification of the DNA)or for expression. Various vectors are publicly available. The vectormay, for example, be in the form of a plasmid, cosmid, viral particle,or phage. The appropriate nucleic acid sequence may be inserted into thevector by a variety of procedures. In general, DNA is inserted into anappropriate restriction endonuclease site(s) using techniques known inthe art. Vector components generally include, but are not limited to,one or more of a signal sequence, an origin of replication, one or moremarker genes, an enhancer element, a promoter, and a transcriptiontermination sequence. Construction of suitable vectors containing one ormore of these components employs standard ligation techniques which areknown to the skilled artisan.

[0303] The PRO may be produced recombinantly not only directly, but alsoas a fusion polypeptide with a heterologous polypeptide, which may be asignal sequence or other polypeptide having a specific cleavage site atthe N-terminus of the mature protein or polypeptide. In general, thesignal sequence may be a component of the vector, or it may be a part ofthe PRO-encoding DNA that is inserted into the vector. The signalsequence may be a prokaryotic signal sequence selected, for example,from the group of the alkaline phosphatase, penicillinase, lpp, orheat-stable enterotoxin II leaders. For yeast secretion the signalsequence may be, e.g., the yeast invertase leader, alpha factor leader(including Saccharomyces and Kluyveromyces α-factor leaders, the latterdescribed in U.S. Pat. No. 5,010,182), or acid phosphatase leader, theC. albicans glucoamylase leader (EP 362,179 published Apr. 4, 1990), orthe signal described in WO 90/13646 published Nov. 15, 1990. Inmammalian cell expression, mammalian signal sequences may be used todirect secretion of the protein, such as signal sequences from secretedpolypeptides of the same or related species, as well as viral secretoryleaders.

[0304] Both expression and cloning vectors contain a nucleic acidsequence that enables the vector to replicate in one or more selectedhost cells. Such sequences are well known for a variety of bacteria,yeast, and viruses. The origin of replication from the plasmid pEBR322is suitable for most Gram-negative bacteria, the 2μ plasmid origin issuitable for yeast, and various viral origins (SV40, polyoma,adenovirus, VSV or BPV) are useful for cloning vectors in mammaliancells.

[0305] Expression and cloning vectors will typically contain a selectiongene, also termed a selectable marker. Typical selection genes encodeproteins that (a) confer resistance to antibiotics or other toxins,e.g., ampicillin, neomycin, methotrexate, or tetracycline, (b)complement auxotrophic deficiencies, or (c) supply critical nutrientsnot available from complex media, e.g., the gene encoding D-alanineracemase for Bacilli.

[0306] An example of suitable selectable markers for mammalian cells arethose that enable the identification of cells competent to take up thePRO-encoding nucleic acid, such as DHFR or thymidine kinase. Anappropriate host cell when wild-type DHFR is employed is the CHO cellline deficient in DHFR activity, prepared and propagated as described byUrlaub et al., Proc. Natl. Acad. Sci. USA, 77:4216 (1980). A suitableselection gene for use in yeast is the trp 1 gene present in the yeastplasmid YRp7 [Stinchcomb et al., Nature, 282:39 (1979); Kingsman et al.,Gene, 7:141 (1979); Tschemper et al., Gene, 10:157 (1980)]. The trp 1gene provides a selection marker for a mutant strain of yeast lackingthe ability to grow in tryptophan, for example, ATCC No. 44076 or PEP4-1[ones, Genetics, 85:12 (1977)].

[0307] Expression and cloning vectors usually contain a promoteroperably linked to the PRO-encoding nucleic acid sequence to direct mRNAsynthesis. Promoters recognized by a variety of potential host cells arewell known. Promoters suitable for use with prokaryotiic hosts includethe β-lactamase and lactose promoter systems [Chang et al., Nature,275:615 (1978); Goeddel et al., Nature, 281:544 (1979)], alkalinephosphatase, a tryptophan (trp) promoter system [Goeddel, Nucleic AcidsRes., 8:4057 (1980); EP 36,776], and hybrid promoters such as the tacpromoter [deBoer et al., Proc. Natl. Acad. Sci. USA, 80:21-25 (1983)].Promoters for use in bacterial systems also will contain aShine-Dalgarno (S.D.) sequence operably linked to the DNA encoding PRO.

[0308] Examples of suitable promoting sequences for use with yeast hostsinclude the promoters for 3-phosphoglycerate kinase [Hitzeman et al., J.Biol. Chem., 255:2073 (1980)] or other glycolytic enzymes [Hess et al.,J. Adv. Enzyme Reg., 7:149 (1968); Holland, Biochemistry, 17:4900(1978)], such as enolase, glyceraldehyde-3-phosphate dehydrogenase,hexokinase, pyruvate decarboxylase, phosphofructokinase,glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvatekinase, triosephosphate isomerase, phosphoglucose isomerase, andglucokinase.

[0309] Other yeast promoters, which are inducible promoters having theadditional advantage of transcription controlled by growth conditions,are the promoter regions for alcohol dehydrogenase 2, isocytochrome C,acid phosphatase, degradative enzymes associated with nitrogenmetabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase,and enzymes responsible for maltose and galactose utilization. Suitablevectors and promoters for use in yeast expression are further describedin EP 73,657.

[0310] PRO transcription from vectors in mammalian host cells iscontrolled, for example, by promoters obtained from the genomes ofviruses such as polyoma virus, fowipox virus (UK 2,211,504 publishedJul. 5, 1989), adenovirus (such as Adenovirus 2), bovine papillomavirus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis-Bvirus and Simian Virus 40 (SV40), from heterologous mammalian promoters,e.g., the actin promoter or an immunoglobulin promoter, and fromheat-shock promoters, provided such promoters are compatible with thehost cell systems.

[0311] Transcription of a DNA encoding the PRO by higher eukaryotes maybe increased by inserting an enhancer sequence into the vector.Enhancers are cis-acting elements of DNA, usually about from 10 to 300bp, that act on a promoter to increase its transcription. Many enhancersequences are now known from mammalian genes (globin, elastase, albumin,α-fetoprotein, and insulin). Typically, however, one will use anenhancer from a eukaryotic cell virus. Examples include the SV40enhancer on the late side of the replication origin (bp 100-270), thecytomegalovirus early promoter enhancer, the polyoma enhancer on thelate side of the replication origin, and adenov/irus enhancers. Theenhancer may be spliced into the vector at a position 5′ or 3′ to thePRO coding sequence, but is preferably located at a site 5′ from thepromoter.

[0312] Expression vectors used in eukarvotic host cells (yeast, fungi,insect, plant, animal, human, or nucleated cells from othermulticellular organisms) will also contain sequences necessary for thetermination of transcription and for stabilizing the mRNA. Suchsequences are commonly available from the 5′ and, occasionally 3′,untranslated regions of eukaryotic or viral DNAs or cDNAs. These regionscontain nucleotide segments transcribed as polyadenylated fragments inthe untranslated portion of the mRNA encoding PRO.

[0313] Still other methods, vectors, and host cells suitable foradaptation to the synthesis of PRO in recombinant vertebrate cellculture are described in Gething et al., Nature, 293:620-625 (1981);Mantei et al., Nature, 281:40-46 (1979); EP 117,060; and EP 117,058.

[0314] 4. Detecting Gene Amplification/Expression

[0315] Gene amplification and/or expression may be measured in a sampledirectly, for example, by conventional Southern blotting, Northernblotting to quantitate the transcription of mRNA [Thomas, Proc. Natl.Acad. Sci. USA, 77:5201(1980)], dot blotting (DINA analysis), or in situhybridization, using an appropriately labeled probe, based on thesequences provided herein. Alternatively, antibodies may be employedthat can recognize specific duplexes, including DNA duplexes, RNAduplexes, and DNA-RNA hybrid duplexes or DNA-protein duplexes. Theantibodies in turn may be labeled and the assay may be carried out wherethe duplex is bound to a surface, so that upon the formation of duplexon the surface, the presence of antibody bound to the duplex can bedetected.

[0316] Gene expression, alternatively, may be measured by immunologicalmethods, such as immunohistochemical staining of cells or tissuesections and assay of cell culture or body fluids, to quantitatedirectly the expression of gene product. Antibodies useful forimmunohistochemical staining and/or assay of sample fluids may be eithermonoclonal or polyclonal, and may be prepared in any mammal.Conveniently, the antibodies may be prepared against a native sequencePRO polypeptide or against a synthetic peptide based on the DNAsequences provided herein or against exogenous sequence fused to PRO DNAand encoding a specific antibody epitope.

[0317] 5. Purification of Polypeptide

[0318] Forms of PRO may be recovered from culture medium or from hostcell lysates. If membrane-bound, it can be released from the membraneusing a suitable detergent solution (e.g. Triton-X 100) or by enzymaticcleavage. Cells employed in expression of PRO can be disrupted byvarious physical or chemical means, such as freeze-thaw cycling,sonication, mechanical disruption, or cell lysing agents.

[0319] It may be desired to purify PRO from recombinant cell proteins orpolypeptides. The following procedures are exemplary of suitablepurification procedures: by fractionation on an ion-exchange column;ethanol precipitation; reverse phase HPLC; chromatography on silica oron a cation-exchange resin such as DEAE; chromatofocusing; SDS-PAGE;ammonium sulfate precipitation; gel filtration using, for example,Sephadex G-75; protein A Sepharose columns to remove contaminants suchas IgG; and metal chelating columns to bind epitope-tagged forms of thePRO. Various methods of protein purification may be employed and suchmethods are known in the art and described for example in Deutscher,Methods in Enzymology, 182 (1990); Scopes, Protein Purification:Principles and Practice, Springer-Verlag, New York (1982). Thepurification step(s) selected will depend, for example, on the nature ofthe production process used and the particular PRO produced.

[0320] E. Uses for PRO

[0321] Nucleotide sequences (or their complement) encoding PRO havevarious applications in the art of molecular biology, including uses ashybridization probes, in chromosome and gene mapping and in thegeneration of anti-sense RNA and DNA. PRO nucleic acid will also beuseful for the preparation of PRO polypeptides by the recombinanttechniques described herein.

[0322] The full-length native sequence PRO gene, or portions thereof,may be used as hybridization probes for a cDNA library to isolate thefull-length PRO cDNA or to isolate still other cDNAs (for instance,those encoding naturally-occurring variants of PRO or PRO from otherspecies) which have a desired sequence identity to the native PROsequence disclosed herein. Optionally, the length of the probes will beabout 20 to about 50 bases. The hybridization probes may be derived fromat least partially novel regions of the full length native nucleotidesequence wherein those regions may be determined without undueexperimentation or from genomic sequences including promoters, enhancerelements and introns of native sequence PRO. By way of example, ascreening method will comprise isolating the coding region of the PROgene using the known DNA sequence to synthesize a selected probe ofabout 40 bases. Hybridization probes may be labeled by a variety oflabels, including radionucleotides such as ³²P or ³⁵S, or enzymaticlabels such as alkaline phosphatase coupled to the probe viaavidin/biotin coupling systems. Labeled probes having a sequencecomplementary to that of the PRO gene of the present invention can beused to screen libraries of human cDNA, genomic DNA or mRNA to determinewhich members of such libraries the probe hybridizes to. Hybridizationtechniques are described in further detail in the Examples below.

[0323] Any EST sequences disclosed in the present application maysimilarly be employed as probes, using the methods disclosed herein.

[0324] Other useful fragments of the PRO nucleic acids include antisenseor sense oligonucleotides comprising a singe-stranded nucleic acidsequence (either RNA or DNA) capable of binding to target PRO mRNA(sense) or PRO DNA (antisense) sequences. Antisense or senseoligonucleotides, according to the present invention, comprise afragment of the coding region of PRO DNA. Such a fragment generallycomprises, at least about 14 nucleotides, preferably from about 14 to 30nucleotides. The ability to derive an antisense or a senseoligonucleotide, based upon a cDNA sequence encoding a given protein isdescribed in, for example, Stein and Cohen (Cancer Res. 48:2659, 1988)and van der Krol et al. (BioTechniques 6:958, 1988).

[0325] Binding of antisense or sense oligonucleotides to target nucleicacid sequences results in the formation of duplexes that blocktranscription or translation of the target sequence by one of severalmeans, including enhanced degradation of the duplexes, prematuretermination of transcription or translation, or by other means. Theantisense oligonucleotides thus may be used to block expression of PROproteins. Antisense or sense oligonucleotides further compriseoligonucleotides having modified sugar-phosphodiester backbones (orother sugar linkages, such as those described in WO 91/06629) andwherein such sugar linkages are resistant to endogenous nucleases. Sucholigonucleotides with resistant sugar linkages are stable in vivo (i.e.,capable of resisting enzymatic degradation) but retain sequencespecificity to be able to bind to target nucleotide sequences.

[0326] Other examples of sense or antisense oligonucleotides includethose oligonucleotides which are covalently linked to organic moieties,such as those described in WO 90/10048, and other moieties thatincreases affinity of the oligonucleotide for a target nucleic acidsequence, such as poly-(L-lysine). Further still, intercalating agents,such as ellipticine, and alkylating agents or metal complexes may beattached to sense or antisense oligonucleotides to modify bindingspecificities of the antisense or sense oligonucleotide for the targetnucleotide sequence.

[0327] Antisense or sense oligonucleotides may be introduced into a cellcontaining the target nucleic acid sequence by any gene transfer method,including, for example, CaPO⁴⁻ mediated DNA transfection,electroporation, or by using gene transfer vectors such as Epstein-Barrvirus. In a preferred procedure, an antisense or sense oligonucleotideis inserted into a suitable retroviral vector. A cell containing thetarget nucleic acid sequence is contacted with the recombinantretroviral vector, either in vivo or ex vivo. Suitable retroviralvectors include, but are not limited to, those derived from the murineretrovirus M-MuLV, N2 (a retrovirus derived from M-MuLV), or the doublecopy vectors designated DCT5A, DCT5B and DCT5C (see WO 90/13641).

[0328] Sense or antisense oligonucleotides also may be introduced into acell containing the target nucleotide sequence by formation of aconjugate with a ligand binding molecule, as described in WO 91/04753.Suitable ligand binding molecules include, but are not limited to, cellsurface receptors, growth factors, other cytokines, or other ligandsthat bind to cell surface receptors. Preferably, conjugation of theligand binding molecule does not substantially interfere with theability of the ligand binding molecule to bind to its correspondingmolecule or receptor, or block entry of the sense or antisenseoligonucleotide or its conjugated version into the cell.

[0329] Alternatively, a sense or an antisense oligonucleotide may beintroduced into a cell containing the target nucleic acid sequence byformation of an oligonucleotide-lipid complex, as described in WO90/10448. The sense or antisense oligonucleotide-lipid complex ispreferably dissociated within the cell by an endogenous lipase.

[0330] Antisense or sense RNA or DNA molecules are generally at leastabout 5 bases in length, about 10 bases in length, about 15 bases inlength, about 20 bases in length, about 25 bases in length, about 30bases in length, about 35 bases in length, about 40 bases in length,about 45 bases in length, about 50 bases in length, about 55 bases inlength, about 60 bases in length, about 65 bases in length, about 70bases in length, about 75 bases in length, about 80 bases in length,about 85 bases in length, about 90 bases in length, about 95 bases inlength, about 500 bases in length, or more.

[0331] The probes may also be employed in PCR techniques to generate apool of sequences for identification of closely related PRO codingsequences.

[0332] Nucleotide sequences encoding a PRO can also be used to constructhybridization probes for mapping the gene which encodes that PRO and forthe genetic analysis of individuals with genetic disorders. Thenucleotide sequences provided herein may be mapped to a chromosome andspecific regions of a chromosome using known techniques, such as in situhybridization, linkage analysis against known chromosomal markers, andhybridization screening with libraries.

[0333] When the coding sequences for PRO encode a protein which binds toanother protein (example, where the PRO is a receptor), the PRO can beused in assays to identify the other proteins or molecules involved inthe binding interaction. By such methods, inhibitors of thereceptor/ligand binding interaction can be identified. Proteins involvedin such binding interactions can also be used to screen for peptide orsmall molecule inhibitors or agonists of the binding interaction. Also,the receptor PRO can be used to isolate correlative ligand(s). Screeningassays can be designed to find lead compounds that mimic the biologicalactivity of a native PRO or a receptor for PRO. Such screening assayswill include assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates. Small molecules contemplated include syntheticorganic or inorganic compounds. The assays can be performed in a varietyof formats, including protein-protein binding assays, biochemicalscreening assays, immunoassays and cell based assays, which are wellcharacterized in the art.

[0334] Nucleic acids which encode PRO or its modified forms can also beused to generate either transgenic animals or “knock out” animals which,in turn, are useful in the development and screening of therapeuticallyuseful reagents. A transgenic animal (e.g., a mouse or rat) is an animalhaving cells that contain a transgene, which transgene was introducedinto the animal or an ancestor of the animal at a prenatal, e.g., anembryonic stage. A transgene is a DNA which is integrated into thegenome of a cell from which a transgenic animal develops. In oneembodiment, cDNA encoding PRO can be used to clone genomic DNA encodingPRO in accordance with established techniques and the genomic sequencesused to generate transgenic animals that contain cells which express DNAencoding PRO. Methods for generating transgenic animals, particularlyanimals such as mice or rats, have become conventional in the art andare described, for example, in U.S. Pat. Nos. 4,736,866 and 4,870,009.Typically, particular cells would be targeted for PRO transgeneincorporation with tissue-specific enhancers. Transgenic animals thatinclude a copy of a transgene encoding PRO introduced into the germ lineof the animal at an embryonic stage can be used to examine the effect ofincreased expression of DNA encoding PRO. Such animals can be used astester animals for reagents thought to confer protection from, forexample, pathological conditions associated with its overexpression. Inaccordance with this facet of the invention, an animal is treated withthe reagent and a reduced incidence of the pathological condition,compared to untreated animals bearing the transgene, would indicate apotential therapeutic intervention for the pathological condition.

[0335] Alternatively, non-human homologues of PRO can be used toconstruct a PRO “knock Out” animal which has a defective or altered geneencoding PRO as a result of homologous recombination between theendogenous gene encoding PRO and altered genomic DNA encoding PROintroduced into an embryonic stem cell of the animal. For example, cDNAencoding PRO can be used to clone genomic DNA encoding PRO in accordancewith established techniques. A portion of the genomic DNA encoding PROcan be deleted or replaced with another gene, such as a gene encoding aselectable marker which can be used to monitor integration. Typically,several kilobases of unaltered flanking DNA (both at the 5′ and 3′ ends)are included in the vector [see e.g., Thomas and Capecchi, Cell, 51:503(1987) for a description of homologous recombination vectors]. Thevector is introduced into an embryonic stem cell line (e.g., byelectroporation) and cells in which the introduced DNA has homologouslyrecombined with the endogenous DNA are selected [see e.g., Li et al.,Cell, 69:915 (1992)]. The selected cells are then injected into ablastocyst of an animal (e.g., a mouse or rat) to form aggregationchimeras [see e.g., Bradley, in Teratocarcinomas and Embryonic StemCells: A Practical Approach, E. J. Robertson, ed. (IRL, Oxford, 1987),pp. 113-152]. A chimeric embryo can then be implanted into a suitablepseudopregnant female foster animal and the embryo brought to term tocreate a “knock out” animal. Progeny harboring the homologouslyrecombined DNA in their germ cells can be identified by standardtechniques and used to breed animals in which all cells of the animalcontain the homologously recombined DNA. Knockout animals can becharacterized for instance, for their ability to defend against certainpathological conditions and for their development of pathologicalconditions due to absence of the PRO polypeptide.

[0336] Nucleic acid encoding the PRO polypeptides may also be used ingene therapy. In gene therapy applications, genes are introduced intocells in order to achieve in vivo synthesis of a therapeuticallyeffective genetic product, for example for replacement of a defectivegene. “Gene therapy” includes both conventional gene therapy where alasting effect is achieved by a single treatment, and the administrationof gene therapeutic agents, which involves the one time or repeatedadministration of a therapeutically effective DNA or mRNA. AntisenseRNAs and DNAs can be used as therapeutic agents for blocking theexpression of certain genes in vivo. It has already been shown thatshort antisense oligonucleotides can be imported into cells where theyact as inhibitors, despite their low intracellular concentrations causedby their restricted uptake by the cell membrane. (Zamecnik et al., Proc.Natl. Acad. Sci. USA 83:4143-4146[1986]). The oligonucleotides can bemodified to enhance their uptake, e.g. by substituting their negativelycharged phosphodiester groups by uncharged groups.

[0337] There are a variety of techniques available for introducingnucleic acids into viable cells. The techniques vary depending uponwhether the nucleic acid is transferred into cultured cells in vitro, orin vivo in the cells of the intended host. Techniques suitable for thetransfer of nucleic acid into mammalian cells in vitro include the useof liposomes, electroporation, microinjection, cell fusion,DEAE-dextran, the calcium phosphate precipitation method, etc. Thecurrently preferred in vivo gene transfer techniques includetransfection with viral (typically retroviral) vectors and viral coatprotein-liposome mediated transfection (Dzau et al., Trends inBiotechnology 11, 205-210[1993]). In some situations it is desirable toprovide the nucleic acid source with an agent that targets the targetcells, such as an antibody specific for a cell surface membrane proteinor the target cell, a ligand for a receptor on the target cell, etc.Where liposomes are employed, proteins which bind to a cell surfacemembrane protein associated with endocytosis may be used for targetingand/or to facilitate uptake, e.g. capsid proteins or fragments thereoftropic for a particular cell type, antibodies for proteins which undergointernalization in cycling, proteins that target intracellularlocalization and enhance intracellular half-life. The technique ofreceptor-mediated endocytosis is described, for example, by Wu et al.,J. Biol. Chem. 262, 4429-4432 (1987); and Wagner et al., Proc. Natl.Acad. Sci. USA 87, 3410-3414 (1990). For review of gene marking and genetherapy protocols see Anderson et al., Science 256, 808-813 (1992).

[0338] The PRO polypeptides described herein may also be employed asmolecular weight markers for protein electrophoresis purposes and theisolated nucleic acid sequences may be used for recombinantly expressingthose markers.

[0339] The nucleic acid molecules encoding the PRO polypeptides orfragments thereof described herein are useful for chromosomeidentification. In this regard, there exists an ongoing need to identifynew chromosome markers, since relatively few chromosome markingreagents, based upon actual sequence data are presently available. EachPRO nucleic acid molecule of the present invention can be used as achromosome marker.

[0340] The PRO polypeptides and nucleic acid molecules of the presentinvention may also be used diagnostically for tissue typing, wherein thePRO polypeptides of the present invention may be differentiallyexpressed in one tissue as compared to another, preferably in a diseasedtissue as compared to a normal tissue of the same tissue type. PROnucleic acid molecules will find use for generating probes for PCR,Northern analysis, Southern analysis and Western analysis.

[0341] The PRO polypeptides described herein may also be employed astherapeutic agents. The PRO polypeptides of the present invention can beformulated according to known methods to prepare pharmaceutically usefulcompositions, whereby the PRO product hereof is combined in admixturewith a pharmaceutically acceptable carrier vehicle. Therapeuticformulations are prepared for storage by mixing the active ingredienthaving the desired degree of purity with optional physiologicallyacceptable carriers, excipients or stabilizers (Remington'sPharmaceutical Sciences 16th edition, Osol, A. Ed. (1980)), in the formof lyophilized formulations or aqueous solutions.

[0342] Acceptable carriers, excipients or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate and other organic acids; antioxidantsincluding ascorbic acid; low molecular weight (less than about 10residues) polypeptides; proteins, such as serum albumin, gelatin orimmunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone,amino acids such as glycine, glutamine, asparagine, arginine or lysine;monosaccharides, disaccharides and other carbohydrates includingglucose, mannose, or dextrins; chelating agents such as EDTA; sugaralcohols such as mannitol or sorbitol; salt-forming counterions such assodium; and/or nonionic surfactants such as TWEEN™, PLURONICS™ or PEG.

[0343] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes, prior to or following lyophilization andreconstitution.

[0344] Therapeutic compositions herein generally are placed into acontainer having a sterile access port, for example, an intravenoussolution bag or vial having a stopper pierceable by a hypodermicinjection needle.

[0345] The route of administration is in accord with known methods, e.g.injection or infusion by intravenous, intraperitoneal, intracerebral,intramuscular, intraocular, intraarterial or intralesional routes,topical administration, or by sustained release systems.

[0346] Dosages and desired drug concentrations of pharmaceuticalcompositions of the present invention may vary depending on theparticular use envisioned. The determination of the appropriate dosageor route of administration is well within the skill of an ordinaryphysician. Animal experiments provide reliable guidance for thedetermination of effective doses for human therapy. Interspecies scalingof effective doses can be performed following the principles laid downby Mordenti, J. and Chappell, W. “The use of interspecies scaling intoxicokinetics” In Toxicokinetics and New Drug Development, Yacobi etal., Eds., Pergamon Press, New York 1989, pp. 42-96.

[0347] When in vivo administration of a PRO polypeptide or agonist orantagonist thereof is employed, normal dosage amounts may vary fromabout 10 ng/kg to up to 100 mg/kg of mammal body weight or more per day,preferably about 1 μg/kg/day to 10 mg/kg/day, depending upon the routeof administration. Guidance as to particular dosages and methods ofdelivery is provided in the literature; see, for example, U.S. Pat. Nos.4,657,760; 5,206,344; or 5,225,212. It is anticipated that differentformulations will be effective for different treatment compounds anddifferent disorders, that administration targeting one organ or tissue,for example, may necessitate delivery in a manner different from that toanother organ or tissue.

[0348] Where sustained-release administration of a PRO polypeptide isdesired in a formulation with release characteristics suitable for thetreatment of any disease or disorder requiring administration of the PROpolypeptide, microencapsulation of the PRO polypeptide is contemplated.Microencapsulation of recombinant proteins for sustained release hasbeen successfully performed with human growth hormone (rhGH),interferon-(rhlFN-), interleukin-2, and MN rgp120. Johnson et al., Nat.Med., 2:795-799 (1996); Yasuda, Biomed. Ther., 27:1221-1223 (1993); Horaet al., Bio/Technology, 8:755-758 (1990); Cleland, “Design andProduction of Single Immunization Vaccines Using PolylactidePolyglycolide Microsphere Systems,” in Vaccine Design: The Subunit andAdjuvant Approach, Powell and Newman, eds, (Plenum Press: New York,1995), pp. 439-462; WO 97/03692, WO 96/40072, WO 96/07399; and U.S. Pat.No. 5,654,010.

[0349] The sustained-release formulations of these proteins weredeveloped using poly-lactic-coglycolic acid (PLGA) polymer due to itsbiocompatibility and wide range of biodegradable properties. Thedegradation products of PLGA, lactic and glycolic acids, can be clearedquickly within the human body. Moreover, the degradability of thispolymer can be adjusted from months to years depending on its molecularweight and composition. Lewis, “Controlled release of bioactive agentsfrom lactide/glycolide polymer,” in: M. Chasin and R. Langer (Eds.),Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: NewYork, 1990), pp.1-41.

[0350] This invention encompasses methods of screening compounds toidentify those that mimic the PRO polypeptide (agonists) or prevent theeffect of the PRO polypeptide (antagonists). Screening assays forantagonist drug candidates are designed to identify compounds that bindor complex with the PRO polypeptides encoded by the genes identifiedherein, or otherwise interfere with the interaction of the encodedpolypeptides with other cellular proteins. Such screening assays willinclude assays amenable to high-throughput screening of chemicallibraries, making them particularly suitable for identifying smallmolecule drug candidates.

[0351] The assays can be performed in a variety of formats, includingprotein-protein binding assays, biochemical screening assays,immunoassays, and cell-based assays, which are well characterized in theart.

[0352] All assays for antagonists are common in that they call forcontacting the drug candidate with a PRO polypeptide encoded by anucleic acid identified herein under conditions and for a timesufficient to allow these two components to interact.

[0353] In binding assays, the interaction is binding and the complexformed can be isolated or detected in the reaction mixture. In aparticular embodiment, the PRO polypeptide encoded by the geneidentified herein or the drug candidate is immobilized on a solid phase,e.g., on a microtiter plate, by covalent or non-covalent attachments.Non-covalent attachment generally is accomplished by coating the solidsurface with a solution of the PRO polypeptide and drying.Alternatively, an immobilized antibody, e.g., a monoclonal antibody,specific for the PRO polypeptide to be immobilized can be used to anchorit to a solid surface. The assay is performed by adding thenon-immobilized component, which may be labeled by a detectable label,to the immobilized component, e.g., the coated surface containing theanchored component. When the reaction is complete, the non-reactedcomponents are removed, e.g., by washing, and complexes anchored on thesolid surface are detected. When the originally non-immobilizedcomponent carries a detectable label, the detection of label immobilizedon the surface indicates that complexing occurred. Where the originallynon-immobilized component does not carry a label, complexing can bedetected, for example, by using a labeled antibody specifically bindingthe immobilized complex.

[0354] If the candidate compound interacts with but does not bind to aparticular PRO polypeptide encoded by a gene identified herein, itsinteraction with that polypeptide can be assayed by methods well knownfor detecting protein-protein interactions. Such assays includetraditional approaches, such as, e.g., cross-linking,co-immunoprecipitation, and co-purification through gradients orchromatographic columns. In addition, protein-protein interactions canbe monitored by using a yeast-based genetic system described by Fieldsand co-workers (Fields and Song, Nature (London), 340:245-246 (1989);Chien et al., Proc. Natl. Acad. Sci. USA, 88:9578-9582 (1991)) asdisclosed by Chevray and Nathans, Proc. Natl. Acad. Sci. USA, 89:5789-5793 (1991). Many transcriptional activators, such as yeast GAL4,consist of two physically discrete modular domains, one acting as theDNA-binding domain, the other one functioning as thetranscription-activation domain. The yeast expression system describedin the foregoing publications (generally referred to as the “two-hybridsystem”) takes advantage of this property, and employs two hybridproteins, one in which the target protein is fused to the DNA-bindingdomain of GAL4, and another, in which candidate activating proteins arefused to the activation domain. The expression of a GAL1-lacZ reportergene under control of a GAL4-activated promoter depends onreconstitution of GAL4 activity via protein-protein interaction.Colonies containing interacting polypeptides are detected with achromogenic substrate for β-galactosidase. A complete kit (MATCHMAKER™)for identifying protein-protein interactions between two specificproteins using the two-hybrid technique is commercially available fromClontech. This system can also be extended to map protein domainsinvolved in specific protein interactions as well as to pinpoint aminoacid residues that are crucial for these interactions.

[0355] Compounds that interfere with the interaction of a gene encodinga PRO polypeptide identified herein and other intra- or extracellularcomponents can be tested as follows: usually a reaction mixture isprepared containing the product of the gene and the intra- orextracellular component under conditions and for a time allowing for theinteraction and binding of the two products. To test the ability of acandidate compound to inhibit binding, the reaction is run in theabsence and in the presence of the test compound. In addition, a placebomay be added to a third reaction mixture, to serve as positive control.The binding (complex formation) between the test compound and the intra-or extracellular component present in the mixture is monitored asdescribed hereinabove. The formation of a complex in the controlreaction(s) but not in the reaction mixture containing the test compoundindicates that the test compound interferes with the interaction of thetest compound and its reaction partner.

[0356] To assay for antagonists, the PRO polypeptide may be added to acell along with the compound to be screened for a particular activityand the ability of the compound to inhibit the activity of interest inthe presence of the PRO polypeptide indicates that the compound is anantagonist to the PRO polypeptide. Alternatively, antagonists may bedetected by combining the PRO polypeptide and a potential antagonistwith membrane-bound PRO polypeptide receptors or recombinant receptorsunder appropriate conditions for a competitive inhibition assay. The PROpolypeptide can be labeled, such as by radioactivity, such that thenumber of PRO polypeptide molecules bound to the receptor can be used todetermine the effectiveness of the potential antagonist. The geneencoding the receptor can be identified by numerous methods known tothose of skill in the art, for example, ligand panning and FACS sorting.Coligan et al., Current Protocols in Immun., 1(2): Chapter 5 (1991).Preferably, expression cloning is employed wherein polyadenylated RNA isprepared from a cell responsive to the PRO polypeptide and a cDNAlibrary created from this RNA is divided into pools and used totransfect COS cells or other cells that are not responsive to the PROpolypeptide. Transfected cells that are grown on glass slides areexposed to labeled PRO polypeptide. The PRO polypeptide can be labeledby a variety of means including iodination or inclusion of a recognitionsite for a site-specific protein kinase. Following fixation andincubation, the slides are subjected to autoradiographic analysis.Positive pools are identified and sub-pools are prepared andre-transfected using an interactive sub-pooling and re-screeningprocess, eventually yielding a single clone that encodes the putativereceptor.

[0357] As an alternative approach for receptor identification, labeledPRO polypeptide can be photoaffinity-linked with cell membrane orextract preparations that express the receptor molecule. Cross-linkedmaterial is resolved by PAGE and exposed to X-ray film. The labeledcomplex containing the receptor can be excised, resolved into peptidefragments, and subjected to protein micro-sequencing. The amino acidsequence obtained from micro-sequencing would be used to design a set ofdegenerate oligonucleotide probes to screen a cDNA library to identifythe gene encoding the putative receptor.

[0358] In another assay for antagonists, mammalian cells or a membranepreparation expressing the receptor would be incubated with labeled PROpolypeptide in the presence of the candidate compound. The ability ofthe compound to enhance or block this interaction could then bemeasured.

[0359] More specific examples of potential antagonists include anoligonucleotide that binds to the fusions of immunoglobulin with PROpolypeptide, and, in particular, antibodies including, withoutlimitation, poly- and monoclonal antibodies and antibody fragments,single-chain antibodies, anti-idiotypic antibodies, and chimeric orhumanized versions of such antibodies or fragments, as well as humanantibodies and antibody fragments. Alternatively, a potential antagonistmay be a closely related protein, for example, a mutated form of the PROpolypeptide that recognizes the receptor but imparts no effect, therebycompetitively inhibiting the action of the PRO polypeptide.

[0360] Another potential PRO polypeptide antagonist is an antisense RNAor DNA construct prepared using antisense technology, where, e.g., anantisense RNA or DNA molecule acts to block directly the translation ofmRNA by hybridizing to targeted mRNA and preventing protein translation.Antisense technology can be used to control gene expression throughtriple-helix formation or antisense DNA or RNA, both of which methodsare based on binding of a polynucleotide to DNA or RNA. For example, the5′ coding portion of the polynucleotide sequence, which encodes themature PRO polypeptides herein, is used to design an antisense RNAoligonucleotide of from about 10 to 40 base pairs in length. A DNAoligonucleotide is designed to be complementary to a region of the geneinvolved in transcription (triple helix—see Lee et al., Nucl. AcidsRes., 6:3073 (1979); Cooney et al., Science, 241: 456 (1988); Dervan etal., Science, 251:1360 (1991)), thereby preventing transcription and theproduction of the PRO polypeptide. The antisense RNA oligonucleotidehybridizes to the mRNA in vivo and blocks translation of the mRNAmolecule into the PRO polypeptide (antisense—Okano, Neurochem., 56:560(1991); Oligodeoxynucleotides as Antisense Inhibitors of Gene Expression(CRC Press: Boca Raton, Fla., 1988). The oligonucleotides describedabove can also be delivered to cells such that the antisense RNA or DNAmay be expressed in vivo to inhibit production of the PRO polypeptide.When antisense DNA is used, oligodeoxyribonucleotides derived from thetranslation-initiation site, e.g., between about −10 and +10 positionsof the target gene nucleotide sequence, are preferred.

[0361] Potential antagonists include small molecules that bind to theactive site, the receptor binding site, or growth factor or otherrelevant binding site of the PRO polypeptide, thereby blocking thenormal biological activity of the PRO polypeptide. Examples of smallmolecules include, but are not limited to, small peptides orpeptide-like molecules, preferably soluble peptides, and syntheticnon-peptidyl organic or inorganic compounds.

[0362] Ribozymes are enzymatic RNA molecules capable of catalyzing thespecific cleavage of RNA. Ribozymes act by sequence-specifichybridization to the complementary target RNA, followed byendonucleolytic cleavage. Specific ribozyme cleavage sites within apotential RNA target can be identified by known techniques. For furtherdetails see, e.g., Rossi, Current Biology, 4:469-471 (1994), and PCTpublication No. WO 97133551 (published Sep. 18, 1997).

[0363] Nucleic acid molecules in triple-helix formation used to inhibittranscription should be single-stranded and composed ofdeoxynucleotides. The base composition of these oligonucleotides isdesigned such that it promotes triple-helix formation via Hoogsteenbase-pairing rules, which generally require sizeable stretches ofpurines or pyrimidines on one strand of a duplex. For further detailssee, e.g., PCT publication No. WO 97/33551, supra.

[0364] These small molecules can be identified by any one or more of thescreening assays discussed hereinabove and/or by any other screeningtechniques well known for those skilled in the art.

[0365] Diagnostic and therapeutic uses of the herein disclosed moleculesmay also be based upon the positive functional assay hits disclosed anddescribed below.

[0366] F. Anti-PRO Antibodies

[0367] The present invention further provides anti-PRO antibodies.Exemplary antibodies include polyclonal, monoclonal, humanized,bispecific, and heteroconjugate antibodies.

[0368] 1. Polyclonal Antibodies

[0369] The anti-PRO antibodies may comprise polyclonal antibodies.Methods of preparing polyclonal antibodies are known to the skilledartisan. Polyclonal antibodies can be raised in a mammal, for example,by one or more injections of an immunizing agent and, if desired, anadjuvant. Typically, the immunizing agent and/or adjuvant will beinjected in the mammal by multiple subcutaneous or intraperitonealinjections. The immunizing agent may include the PRO polypeptide or afusion protein thereof. It may be useful to conjugate the immunizingagent to a protein known to be immunogenic in the mammal beingimmunized. Examples of such immunogenic proteins include but are notlimited to keyhole limpet hemocyanin, serum albumin, bovinethyroglobulin, and soybean trypsin inhibitor. Examples of adjuvantswhich may be employed include Freund's complete adjuvant and MPL-TDMadjuvant (monophosphoryl Lipid A, synthetic trehalose dicorynomycolate).The immunization protocol may be selected by one skilled in the artwithout undue experimentation.

[0370] 2. Monodconal Antibodies

[0371] The anti-PRO antibodies may, alternatively, be monoclonalantibodies. Monoclonal antibodies may be prepared using hybridomamethods, such as those described by Kohler and Milstein, Nature, 256:495(1975). In a hybridoma method, a mouse, hamster, or other appropriatehost animal, is typically immunized with an immunizing agent to elicitlymphocytes that produce or are capable of producing antibodies thatwill specifically bind to the immunizing agent. Alternatively, thelymphocytes may be immunized in vitro.

[0372] The immunizing agent will typically include the PRO polypeptideor a fusion protein thereof. Generally, either peripheral bloodlymphocytes (“PBLs”) are used if cells of human origin are desired, orspleen cells or lymph node cells are used if non-human mammalian sourcesare desired. The lymphocytes are then fused with an immortalized cellline using a suitable fusing agent, such as polyethylene glycol, to forma hybridoma cell [Goding, Monoclonal Antibodies: Principles andPractice, Academic Press, (1986) pp. 59-103]. Immortalized cell linesare usually transformed mammalian cells, particularly myeloma cells ofrodent, bovine and human origin.

[0373] Usually, rat or mouse myeloma cell lines are employed. Thehybridoma cells may be cultured in a suitable culture medium thatpreferably contains one or more substances that inhibit the growth orsurvival of the unfused, immortalized cells. For example, if theparental cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomastypically will include hypoxanthine, aminopterin, and thymidine (“HATmedium”), which substances prevent the growth of HGPRT-deficient cells.

[0374] Preferred immortalized cell lines are those that fuseefficiently, support stable high level expression of antibody by theselected antibody-producing cells, and are sensitive to a medium such asHAT medium. More preferred immortalized cell lines are murine myelomalines, which can be obtained, for instance, from the Salk Institute CellDistribution Center, San Diego, Calif. and the American Type CultureCollection, Manassas, Va. Human myeloma and mouse-human heteromyelomacell lines also have been described for the production of humanmonoclonal antibodies [Kozbor, J. Immunol., 133:3001 (1984); Brodeur etal., Monoclonal Antibody Production Techniques and Applications, MarcelDekker, Inc., New York, (1987) pp. 51-63].

[0375] The culture medium in which the hybridoma cells are cultured canthen be assayed for the presence of monoclonal antibodies directedagainst PRO. Preferably, the binding specificity of monoclonalantibodies produced by the hybridoma cells is determined byimmunoprecipitation or by an in vitro binding assay, such asradioimmunoassay (RIA) or enzyme-linked immunoabsorbent assay (ELISA).Such techniques and assays are known in the art. The binding affinity ofthe monoclonal antibody can, for example, be determined by the Scatchardanalysis of Munson and Pollard, Anal. Biochem., 107:220 (1980).

[0376] After the desired hybridoma cells are identified, the clones maybe subcloned by limiting dilution procedures and grown by standardmethods [Goding, supra]. Suitable culture media for this purposeinclude, for example, Dulbecco's Modified Eagle's Medium and RPMI-L 640medium. Alternatively, the hybridoma cells may be grown in vivo asascites in a mammal.

[0377] The monoclonal antibodies secreted by the subclones may beisolated or purified from the culture medium or ascites fluid byconventional immunoglobulin purification procedures such as, forexample, protein A-Sepharose, hydroxylapatite chromatography, gelelectrophoresis, dialysis, or affinity chromatography.

[0378] The monoclonal antibodies may also be made by recombinant DNAmethods, such as those described in U.S. Pat. No. 4,816,567. DNAencoding the monoclonal antibodies of the invention can be readilyisolated and sequenced using conventional procedures (e.g., by usingoligonucleotide probes that are capable of binding specifically to genesencoding the heavy and light chains of murine antibodies). The hybridomacells of the invention serve as a preferred source of such DNA. Onceisolated, the DNA may be placed into expression vectors, which are thentransfected into host cells such as simian COS cells, Chinese hamsterovary (CHO) cells, or myeloma cells that do not otherwise produceimmunoglobulin protein, to obtain the synthesis of monoclonal antibodiesin the recombinant host cells. The DNA also may be modified, forexample, by substituting the coding sequence for human heavy and lightchain constant domains in place of the homologous murine sequences [U.S.Pat. No. 4,816,567; Morrison et al., supra] or by covalentlyjoining tothe immunoglobulin coding sequence all or part of the coding sequencefor a non-immunoglobulin polypeptide. Such a non-immunoglobulinpolypeptide can be substituted for the constant domains of an antibodyof the invention, or can be substituted for the variable domains of oneantigen-combining site of an antibody of the invention to create achimeric bivalent antibody.

[0379] The antibodies may be monovalent antibodies. Methods forpreparing monovalent antibodies are well known in the art. For example,one method involves recombinant expression of immunoglobulin light chainand modified heavy chain. The heavy chain is truncated generally at anypoint in the Fc region so as to prevent heavy chain crosslinking.Alternatively, the relevant cysteine residues are substituted withanother amino acid residue or are deleted so as to prevent crosslinking.

[0380] In vitro methods are also suitable for preparing monovalentantibodies. Digestion of antibodies to produce fragments thereof,particularly, Fab fragments, can be accomplished using routinetechniques known in the art.

[0381] 3. Human and Humanized Antibodies

[0382] The anti-PRO antibodies of the invention may further comprisehumanized antibodies; or human antibodies. Humanized forms of non-human(e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulinchains or fragments thereof (such as Fv, Fab, Fab′, F(ab′)₂ or otherantigen-binding subsequences of antibodies) which contain minimalsequence derived from non-human immunoglobulin. Humanized antibodiesinclude human immunoglobulins (recipient antibody) in which residuesfrom a complementary determining region (CDR) of the recipient arereplaced by residues from a CDR of a non-human species (donor antibody)such as mouse, rat or rabbit having the desired specificity, affinityand capacity. In some instances, Fv framework residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Humanized antibodies may also comprise residues which are found neitherin the recipient antibody nor in the imported CDR or frameworksequences. In general, the humanized antibody will comprisesubstantially all of at least one, and typically two, variable domains,in which all or substantially all of the CDR regions correspond to thoseof a non-human immunoglobulin and all or substantially all of the FRregions are those of a human immunoglobulin consensus sequence. Thehumanized antibody optimally also will comprise at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin [ones et al., Nature, 321:522-525 (1986); Riechmann etal., Nature, 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol.,2:593-596 (1992)].

[0383] Methods for humanizing non-human antibodies are well known in theart. Generally, a humanized antibody has one or more amino acid residuesintroduced into it from a source which is non-human. These non-humanamino acid residues are often referred to as “import” residues, whichare typically taken from an “import” variable domain. Humanization canbe essentially performed following the method of Winter and co-workers[Jones et al., Nature, 321:522-525 (1986); Riechmann et al., Nature,332:323-327 (1988); Verhoeyen et al., Science, 239:1534-1536 (1988)], bysubstituting rodent CDRs or CDR sequences for the correspondingsequences of a human antibody. Accordingly, such “humanized” antibodiesare chimeric antibodies (U.S. Pat. No. 4,816,567), wherein substantiallyless than an intact human variable domain has been substituted by thecorresponding sequence from a non-human species. In practice, humanizedantibodies are typically human antibodies in which some CDR residues andpossibly some FR residues are substituted by residues from analogoussites in rodent antibodies.

[0384] Human antibodies can also be produced using various techniquesknown in the art, including phage display libraries [Hoogenboom andWinter, J. Mol. Biol., 227:381 (1991); Marks et al., J. Mol. Biol.,222:581 (1991)]. The techniques of Cole et al. and Boerner et al. arealso available for the preparation of human monoclonal antibodies (Coleet all., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77(1985) and Boerner et al., J. Immunol., 147(1):86-95 (1991)]. Similarly,human antibodies can be made by introducing of human immunoglobulin lociinto transgenic animals, e.g., mice in which the endogenousimmunoglobulin genes have been partially or completely inactivated. Uponchallenge, human antibody production is observed, which closelyresembles that seen in humans in all respects, including generearrangement, assembly, and antibody repertoire. This approach isdescribed, for example, in U.S. Pat. Nos. 5,545,807; 5,545,806;5,569,825; 5,625,126; 5,633,425; 5,661,016, and in the followingscientific publications: Marks et al., Bio/Technology 10, 779-78:3(1992); Lonberg et al., Nature 368 856-859 (1994); Morrison, Nature 368,812-13 (1994); Fishwild et al., Nature Biotechnology 14, 845-51 (1996);Neuberger, Nature Biotechnology 14, 826 (1996); Lonberg and Huszar,Intern. Rev. Immunol. 13 65-93 (1995).

[0385] The antibodies may also be affinity matured using known selectionand/or mutagenesis methods as described above. Preferred affinitymatured antibodies have an affinity which is five times, more preferably10 times, even more preferably 20 or 30 times greater than the startingantibody (generally murine, humanized or human) from which the maturedantibody is prepared.

[0386] 4. Bispecific Antibodies

[0387] Bispecific antibodies are monoclonal, preferably human orhumanized, antibodies that have binding specificities for at least twodifferent antigens. In the present case, one of the bindingspecificities is for the PRO, the other one is for any other antigen,and preferably for a cell-surface protein or receptor or receptorsubunit.

[0388] Methods for making bispecific antibodies are known in the art.Traditionally, the recombinant production of bispecific antibodies isbased on the co-expression of two immunoglobulin heavy-chain/light-chainpairs, where the two heavy chains have different specificities [Milsteinand Cuello, Nature, 305:537-539 (1983)]. Because of the randomassortment of immunoglobulin heavy and light chains, these hybridomas(quadromas) produce a potential mixture of ten different antibodymolecules, of which only one has the correct bispecific structure. Thepurification of the correct molecule is usually accomplished by affinitychromatography steps. Similar procedures are disclosed in WO 93/08829,published May 13, 1993, and in Traunecker et al., EMBO J., 10:3655-3659(1991).

[0389] Antibody variable domains with the desired binding specificities(antibody-antigen combining sites) can be fused to immunoglobulinconstant domain sequences. The fusion preferably is with animmunoglobulin heavy-chain constant domain, comprising at least part ofthe hinge, CH2, and CH3 regions. It is preferred to have the firstheavy-chain constant region (CH1) containing the site necessary forlight-chain binding present in at least one of the fusions. DNAsencoding the immunoglobulin heavy-chain fusions and, if desired, theimmunoglobulin light chain, are inserted into separate expressionvectors, and are co-transfected into a suitable host organism. Forfurther details of generating bispecific antibodies see, for example,Suresh et al., Methods in Enzymology, 121:210 (1986).

[0390] According to another approach described in WO 96/27011, theinterface between a pair of antibody molecules can be engineered tomaximize the percentage of heterodimers which are recovered fromrecombinant cell culture. The preferred interface comprises at least apart of the CH3 region of an antibody constant domain. In this method,one or more small amino acid side chains from the interface of the firstantibody molecule are replaced with larger side chains (e.g. tyrosine ortryptophan). Compensatory “cavities” of identical or similar size to thelarge side chain (s) are created on the interface of the second antibodymolecule by replacing large amino acid side chains with smaller ones(e.g. alanine or threonine). This provides a mechanism for increasingthe yield of the heterodimer over other unwanted end-products such ashomodimers.

[0391] Bispecific antibodies can be prepared as full length antibodiesor antibody fragments (e.g. F(ab′)₂ bispecific antibodies). Techniquesfor generating bispecific antibodies; from antibody fragments have beendescribed in the literature. For example, bispecific antibodies can beprepared using chemical linkage. Brennan et al., Science 229:81 (1985)describe a procedure wherein intact antibodies are proteolyticallycleaved to generate F(ab′)₂ fragments. These fragments are reduced inthe presence of the dithiol complexing agent sodium arsenite tostabilize vicinal dithiols and prevent intermolecular disulfideformation. The Fab′ fragments generated are then converted tothionitrobenzoate (TNB) derivatives. One of the Fab′-TNB derivatives isthen reconverted to the Fab′-thiol by reduction with mercaptoethylamineand is mixed with an equimolar amount of the other Fab′-TNB derivativeto form the bispecific antibody. The bispecific antibodies produced canbe used as agents for the selective immobilization of enzymes.

[0392] Fab′ fragments may be directly recovered from E. coli andchemically coupled to form bispecific antibodies. Shalaby et al., J.Exp. Med. 175:217(1992) describe the production of a fully humanizedbispecific antibody F(ab′)₂ molecule. Each Fab′ fragment was separatelysecreted from E. coli and subjected to directed chemical coupling invitro to form the bispecific antibody. The bispecific antibody thusformed was able to bind to cells overexpressing the ErbB2 receptor andnormal human T cells, as well as trigger the lytic activity of humancytotoxic lymphocytes against human breast tumor targets.

[0393] Various technique for making and isolating bispecific antibodyfragments directly from recombinant cell culture have also beendescribed. For example, bispecific antibodies have been produced usingleucine zippers. Kostelny et al. J. Immunol. 148 (5):1547-1553(1992).The leucine zipper peptides from the Fos and Jun proteins were linked tothe Fab′ portions of two different antibodies by gene fusion. Theantibody homodimers were reduced at the hinge region to form monomersand then re-oxidized to form the antibody heterodimers. This method canalso be utilized for the production of antibody homodimers. The“diabody” technology described by Hollinger et al., Proc. Natl. Acad.Sci. USA 90:6444-6448(1993) has provided an alternative mechanism formaking bispecific antibody fragments. The fragments comprise aheavy-chain variable domain (V_(H)) connected to a light-chain variabledomain (V_(L)) by a linker which is too short to allow pairing betweenthe two domains on the same chain. Accordingly, the V_(H) and V_(L)domains of one fragment are forced to pair with the complementary V_(L)and V_(H) domains of another fragment, thereby forming twoantigen-binding sites. Another strategy for making bispecific antibodyfragments by the use of single-chain Fv (sFv) dimers has also beenreported. See, Gruber et al., J. Immunol. 152:5368 (1994). Antibodieswith more than two valencies are contemplated. For example, trispecificantibodies can be prepared. Tutt et al. J. Immunol. 147:60 (1991).

[0394] Exemplary bispecific antibodies may bind to two differentepitopes on a given PRO polypeptide herein. Alternatively, an anti-PROpolypeptide arm may be combined with an arm which binds to a triggeringmolecule on a leukocyte such as a T-cell receptor molecule (e.g. CD2,CD3, CD28, or B7), or Fc receptors for IgG (Fcγ R), such as Fcγ RI(CD64), Fcγ RII (CD32) and FcγRIII (CD16) so as to focus cellulardefense mechanisms to the cell expressing the particular PROpolypeptide. Bispecific antibodies may also be used to localizecytotoxic agents to cells which express a particular PRO polypeptide.These antibodies possess a PRO-binding arm and an arm which binds acytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA,or TE TA. Another bispecific antibody of interest binds the PROpolypeptide and further binds tissue factor (TF).

[0395] 5. Heteroconjugate Antibodies

[0396] Heteroconjugate antibodies are also within the scope of thepresent invention. Heteroconjugate antibodies are composed of twocovalently joined antibodies. Such antibodies have, for example, beenproposed to target immune system cells to unwanted cells [U.S. Pat. No.4,676,980], and for treatment of HIV infection [WO 91/00360; WO92/200373; EP 03089]. It is contemplated that the antibodies may beprepared in vitro using known methods in synthetic protein chemistry,including those involving crosslinking agents. For example, immunotoxinsmay be constructed using a disulfide exchange reaction or by forming athioether bond. Examples of suitable reagents for this purpose includeiminothiolate and methyl-4-mercaptobutyrimidate and those disclosed, forexample, in U.S. Pat. No. 4,676,980.

[0397] 6. Effector Function Engineering

[0398] It may be desirable to modify the antibody of the invention withrespect to effector function, so as to enhance, e.g., the effectivenessof the antibody in treating cancer. For example, cysteine residue(s) maybe introduced into the Fc region, thereby allowing interchain disulfidebond formation in this region. The homodimeric antibody thus generatedmay have improved internalization capability and/or increasedcomplement-mediated cell killing and antibody-dependent cellularcytotoxicity (ADCC). See Caron et al., J. Exp Med., 176: 1191-1195(1992) and Shopes, J. Immunol., 148: 2918-2922 (1992). Homodimericantibodies with enhanced anti-tumor activity may also be prepared usingheterobifunctional cross-linkers as described in Wolff et al. CancerResearch, 53: 2560-2565 (1993). Alternatively, an antibody can beengineered that has dual Fc regions and may thereby have enhancedcomplement lysis and ADCC capabilities. See Stevenson et al.,Anti-Cancer Drug Design, 3: 219-230 (1989).

[0399] 1. Immunoconjugates

[0400] The invention also pertains to immunoconjugates comprising anantibody conjugated to a cytotoxic agent such as a chemotherapeuticagent, toxin (e.g., an enzymatically active toxin of bacterial, fungal,plant, or animal origin, or fragments thereof), or a radioactive isotope(i.e., a radioconjugate).

[0401] Chemotherapeutic agents useful in the generation of suchimmunoconjugates have been described above. Enzymatically active toxinsand fragments thereof that can be used include diphtheria A chain,nonbinding active fragments of diphtheria toxin, exotoxin A chain (fromPseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain,alpha-sarcin, Aleurites fordii proteins, dianthin proteins, Phytolacaamericana proteins (PAPI, PAPII, and PAP-S), momordica charantiainhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin,mitogellin, restrictocin, phenomycin, enomycin, and the tricothecenes. Avariety of radionuclides are available for the production ofradioconjugated antibodies. Examples include ²¹²Bi, ¹³¹I, ¹³¹In, ⁹⁰Y,and ¹⁸⁶Re. Conjugates of the antibody and cytotoxic agent are made usinga variety of bifunctional protein-coupling agents such asN-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane(IT), bifunctional derivatives of imidoesters (such as dimethyladipimidate HCL), active esters (such as disuccinimidyl suberate),aldehydes (such as glutareldehyde), bis-azido compounds (such as bis(p-azidobenzoyl) hexanediamine), bis-diazonium derivatives (such asbis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such astolyene 2,6-diisocyanztte), and bis-active fluorine compounds (such as1,5-difluoro-2,4-dinitrobenzene). For example, a ricin immunotoxin canbe prepared as described in Vitetta et al., Science, 238: 1098 (1987).Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent forconjugation of radionucleotide to the antibody. See WO 94/11026.

[0402] In another embodiment, the antibody may be conjugated to a“receptor” (such streptaviclin) for utilization in tumor pretargetingwherein the antibody-receptor conjugate is administered to the patient,followed by removal of unbound conjugate from the circulation using aclearing agent and then administration of a “ligand” (e.g., avidin) thatis conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0403] 8. Immunoliposomes

[0404] The antibodies disclosed herein may also be formulated asimmunoliposomes. Liposomes containing the antibody are prepared bymethods known in the art, such as described in Epstein et al., Proc.Natl. Acad. Sci. USA, 82: 3688 (1985); Hwang et al., Proc. Natl Acad.Sci. USA, 77: 4030 (1980); and U.S. Pat. Nos. 4,485,045 and 4,544,545.Liposomes with enhanced circulation time are disclosed in U.S. Pat. No.5,013,556.

[0405] Particularly useful liposomes can be generated by thereverse-phase evaporation method with a lipid composition comprisingphosphatidylcholine, cholesterol, and PEG-derivatizedphosphatidylethanolamine (PEG-PE). Liposomes are extruded throughfilters of defined pore size to yield liposomes with the desireddiameter. Fab′ fragments of the antibody of the present invention can beconjugated to the liposomes as described in Martin et al., J. Biol.Chem., 257: 286-288 (1982) via a disulfide-interchange reaction. Achemotherapeutic agent (such as Doxorubicin) is optionally containedwithin the liposome. See Gabizon et al., J. National Cancer Inst.,81(19): 1484 (1989).

[0406] 9. Pharmaceutical Compositions of Antibodies

[0407] Antibodies specifically binding a PRO polypeptide identifiedherein, as well as other molecules identified by the screening assaysdisclosed hereinbefore, can be administered for the treatment of variousdisorders in the form of pharmaceutical compositions.

[0408] If the PRO polypeptide is intracellular and whole antibodies areused as inhibitors, internalizing antibodies are preferred. However,lipofections or liposomes can also be used to deliver the antibody, oran antibody fragment, into cells. Where antibody fragments are used, thesmallest inhibitory fragment that specifically binds to the bindingdomain of the target protein is preferred. For example, based upon thevariable-region sequences of an antibody, peptide molecules can bedesigned that retain the ability to bind the target protein sequence.Such peptides can be synthesized chemically and/or produced byrecombinant DNA technology. See, e.g., Marasco et al., Proc. Natl. Acad.Sci. USA, 90: 7889-7893 (1993). The formulation herein may also containmore than one active compound as necessary for the particular indicationbeing treated, preferably those with complementary activities that donot adversely affect each other. Alternatively, or in addition, thecomposition may comprise an agent that enhances its function, such as,for example, a cytotoxic agent, cytokine, chemotherapeutic agent, orgrowth-inhibitory agent. Such molecules are suitably present incombination in amounts that are effective for the purpose intended.

[0409] The active ingredients may also be entrapped in microcapsulesprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsules and poly-(methylmethacylate) microcapsuiles,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles, andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences, supra.

[0410] The formulations to be used for in vivo administration must besterile. This is readily accomplished by filtration through sterilefiltration membranes.

[0411] Sustained-release preparations may be prepared. Suitable examplesof sustained-release preparations include semipermeable matrices ofsolid hydrophobic polymers containing the antibody, which matrices arein the form of shaped articles, e.g., films, or microcapsules. Examplesof sustained-release matrices include polyesters, hydrogels (forexample, poly(2-hydroxyethyl-methacrylate), or poly(vinylalcohol)),polylactides (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acidand γ ethyl-L-glutamate, non-degradable ethylene-vinyl acetate,degradable lactic acid-glycolic acid copolymers such as the LUPRONDEPOT™ (injectable microspheres composed of lactic acid-glycolic acidcopolymer and leuprolide acetate), and poly-D-(−)-3-hydroxybutyric acid.While polymers such as ethylene-vinyl acetate and lactic acid-glycolicacid enable release of molecules for over 100 days, certain hydrogelsrelease proteins for shorter time periods. When encapsulated antibodiesremain in the body for a long time, they may denature or aggregate as aresult of exposure to moisture at 37° C., resulting in a loss ofbiological activity and possible changes in immunogenicity. Rationalstrategies can be devised for stabilization depending on the mechanisminvolved. For example, if the aggregation mechanism is discovered to beintermolecular S-S bond formation through thio-disulfide interchange,stabilization may be achieved by modifying sulfhydryl residues,lyophilizing from acidic solutions, controlling moisture content, usingappropriate additives, and developing specific polymer matrixcompositions.

[0412] G. Uses for Anti-PRO Antibodies

[0413] The anti-PRO antibodies of the invention have various utilities.For example, anti-PRO antibodies may be used in diagnostic assays forPRO, e.g., detecting its expression (and in some cases, differentialexpression) in specific cells, tissues, or serum. Various diagnosticassay techniques known in the art may be used, such as competitivebinding assays, direct or indirect sandwich assays andimmunoprecipitation assays conducted in either heterogeneous orhomogeneous phases [Zola, Monoclonal Antibodies: A Manual of Techniques,CRC Press, Inc. (1987) pp. 147-158]. The antibodies used in thediagnostic assays can be labeled with a detectable moiety. Thedetectable moiety should be capable of producing, either directly orindirectly, a detectable signal. For example, the detectable moiety maybe a radioisotope, such as ³H, ¹⁴C, ³²P, ³⁵S, or ¹²⁵I, a fluorescent orchemiluminescent compound, such as fluorescein isothiocyanate,rhodamine, or luciferin, or an enzyme, such as alkaline phosphatase,beta-galactosidase or horseradish peroxidase. Any method known in theart for conjugating the antibody to the detectable moiety may beemployed, including those methods described by Hunter et al., Nature,144:945 (1962); David et al., Biochemistry, 13:1014 (1974); Pain et al.,J. Immunol. Meth., 40:219 (1981); and Nygren, J. Histochem. andCytochem., 30:407 (1982).

[0414] Anti-PRO antibodies also are useful for the affinity purificationof PRO from recombinant cell culture or natural sources. In thisprocess, the antibodies against PRO are immobilized on a suitablesupport, such as Sephadex resin or filter paper, using methods wellknown in the art. The immobilized antibody then is contacted with asample containing the PRO to be purified, and thereafter the support iswashed with a suitable solvent that will remove substantially all thematerial in the sample except the PRO, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solventthat will release the PRO from the antibody.

[0415] The following examples are offered for illustrative purposesonly, and are not intended to limit the scope of the present inventionin any way.

[0416] All patent and literature references cited in the presentspecification are hereby incorporated by reference in their entirety.

EXAMPLES

[0417] Commercially available reagents referred to in the examples wereused according to manufacturer's instructions unless otherwiseindicated. The source of those cells identified in the followingexamples, and throughout the specification, by ATCC accession numbers isthe American Type Culture Collection, Manassas, Va.

Example 1 Extracellular Domain Homology Screening to Identify NovelPolypeptides and cDNA Encoding Therefor

[0418] The extracellular domain (ECD) sequences (including the secretionsignal sequence, if any) from about 950 known secreted proteins from theSwiss-Prot public database were used to search EST databases. The ESTdatabases included public databases (e.g., Dayhoff, GenBank), andproprietary databases (e.g. LIFESEQ™, Incyte Pharmaceuticals, Palo Alto,Calif.). The search was performed using the computer program BLAST orBLAST-2 (Altschul et al., Methods in Enzymology 266:460-480 (1996)) as acomparison of the ECD protein sequences to a 6 frame translation of theEST sequences. Those comparisons with a BLAST score of 70 (or in somecases 90) or greater that did not encode known proteins were clusteredand assembled into consensus DNA sequences with the program “phrap”(Phil Green, University of Washington, Seattle, Wash.).

[0419] Using this extracellular domain homology screen, consensus DNAsequences were assembled relative to the other identified EST sequencesusing phrap. In addition, the consensus DNA sequences obtained wereoften (but not always) extended using repeated cycles of BLAST orBLAST-2 and phrap to extend the consensus sequence as far as possibleusing the sources of EST sequences discussed above.

[0420] Based upon the consensus sequences obtained as described above,oligonucleotides were then synthesized and used to identify by PCR acDNA library that contained the sequence of interest and for use asprobes to isolate a clone of the full-length coding sequence for a PROpolypeptide. Forward and reverse PCR primers generally range from 20 to30 nucleotides and are often designed to give a PCR product of about100-1000 bp in length. The probe sequences are typically 40-55 bp inlength. In some cases, additional oligonucleotides are synthesized whenthe consensus sequence is greater than about 1-1.5 kbp. In order toscreen several libraries for a full-length clone, DNA from the librarieswas screened by PCR amplification, as per Ausubel et al., CurrentProtocols in Molecular Biology, with the PCR primer pair. A positivelibrary was then used to isolate clones encoding the gene of interestusing the probe oligonucleotide and one of the primer pairs.

[0421] The cDNA libraries used to isolate the cDNA clones wereconstructed by standard methods using commercially available reagentssuch as those from Invitrogen, San Diego, Calif. The cDNA was primedwith oligo dT containing a NotI site, linked with blunt to SalIhemikinased adaptors, cleaved with NotI, sized appropriately by gelelectrophoresis, and cloned in a defined orientation into a suitablecloning vector (such as pRKB or PRKD; pRKSB is a precursor of pRK5D thatdoes not contain the SfiI site; see, Holmes et al., Science,253:1278-1280 (1991)) in the unique XhoI and NotI sites.

Example 2 Isolation of cDNA Clones by Amylase Screening

[0422] 1. Preparation of oligo dT Primed cDNA Library

[0423] mRNA was isolated from a human tissue of interest using reagentsand protocols from Invitrogen, San Diego, Calif. (Fast Track 2). ThisRNA was used to generate an oligo dT primed cDNA library in the vectorpRK5D using reagents and protocols from Life Technologies, Gaithersburg,Md. (Super Script Plasmid System). In this procedure, the doublestranded cDNA was sized to greater than 1000 bp and the SalI/NotITinkered cDNA was cloned into XhoI/NotI cleaved vector. pRK5D is acloning vector that has an sp6 transcription initiation site followed byan SfiI restriction enzyme site preceding the XhoI/NotI cDNA cloningsites.

[0424] 2. Preparation of Random Primed cDNA Library

[0425] A secondary cDNA library was generated in order to preferentiallyrepresent the 5′ ends of the primary cDNA clones. Sp6 RNA was generatedfrom the primary library (described above), and this RNA was used togenerate a random primed cDNA library in the vector pSST-AMY.0 usingreagents and protocols from Life Technologies (Super Script PlasmidSystem, referenced above). In this procedure the double stranded cDNAwas sized to 500-1000 bp, Tinkered with blunt to NotI adaptors, cleavedwith SfiI, and cloned into SfiI/NotI cleaved vector. pSST-AMY.0 is acloning vector that has a yeast alcohol dehydrogenase promoter precedingthe cDNA cloning sites and the mouse amylase sequence (the maturesequence without the secretion signal) followed by the yeast alcoholdehydrogenase terminator, after the cloning sites. Thus, cDNAs clonedinto this vector that are fused in frame with amylase sequence will leadto the secretion of amylase from appropriately transfected yeastcolonies.

[0426] 3. Transformation and Detection

[0427] DNA from the library described in paragraph 2 above was chilledon ice to which was added electrocompetent DH1 OB bacteria (LifeTechnologies, 20 ml). The bacteria and vector mixture was thenelectroporated as recommended by the manufacturer. Subsequently, SOCmedia (Life Technologies, 1 ml) was added and the mixture was incubatedat 37° C. for 30 minutes. The transformants were then plated onto 20standard 150 mm LB plates containing ampicillin and incubated for 16hours (37° C.). Positive colonies were scraped off the plates and theDNA was isolated from the bacterial pellet using standard protocols,e.g. CsCl-gradient. The purified DNA was then carried on to the yeastprotocols below.

[0428] The yeast methods were divided into three categories: (1)Transformation of yeast with the plasmid/cDNA combined vector; (2)Detection and isolation of yeast clones secreting amylase; and (3) PCRamplification of the insert directly from the yeast colony andpurification of the DNA for sequencing and further analysis.

[0429] The yeast strain used was HD56-5A (ATCC-90785). This strain hasthe following genotype: MAT alpha, ura3-52, leu2-3, leu2-112, his3-11,his3-15, MAL⁺, SUC⁺, GAL⁺. Preferably, yeast mutants can be employedthat have deficient post-translational pathways. Such mutants may havetranslocation deficient alleles in sec 71, sec 72, sec 62, withtruncated sec 71 being most preferred. Alternatively, antagonists(including antisense nucleotides and/or ligands) which interfere withthe normal operation of these genes, other proteins implicated in thispost translation pathway (e.g., SEC61p, SEC72p, SEC62p, SEC63p, TDJ1p orSSA1p-4p) or the complex formation of these proteins may also bepreferably employed in combination with the amylase-expressing yeast.

[0430] Transformation was performed based on the protocol outlined byGietz et al., Nucl. Acid. Res., 20:1425 (1992). Transformed cells werethen inoculated from agar into YEPD complex media broth (100 ml) andgrown overnight at 30° C. The YEPD broth was prepared as described inKaiser et al., Methods in Yeast Genetics, Cold Spring Harbor Press, ColdSpring Harbor, N.Y., p. 207 (1994). The overnight culture was thendiluted to about 2×10⁶ cells/ml (approx. OD₆₀₀=0.1) into fresh YEPDbroth (500 ml) and regrown to 1×10⁷ cells/ml (approx. OD₆₀₀=0.4-0.5).

[0431] The cells were then harvested and prepared for transformation bytransfer into GS3 rotor bottles in a Sorval GS3 rotor at 5,000 rpm for 5minutes, the supernatant discarded, and then resuspended into sterilewater, and centrifuged again in 50 ml falcon tubes at 3,500 rpm in aBeckman GS-6KR centrifuge. The supernatant was discarded and the cellswere subsequently washed with LiAc/TE (10 ml, 10 mM Tris-HCl, 1 mM EDTApH 7.5, 100 mM Li₂OOCCH₃), and resuspended into LiAc/TE (2.5 ml).

[0432] Transformation took place by mixing the prepared cells (100 μl)with freshly denatured single stranded salmon testes DNA (LofstrandLabs, Gaithersburg, Md.) and transforming DNA (1 μg, vol.<10 μl) inmicrofuge tubes. The mixture was mixed briefly by vortexing, then 40%PEG/TE (600 μl, 40% polyethylene glycol-4000, 10 mM Tris-HCl, 1 mM EDTA,100 mM Li₂ OOCCH₃, pH 7.5) was added. This mixture was gently mixed andincubated at 30° C. while agitating for 30 minutes. The cells were thenheat shocked at 42° C. for 15 minutes, and the reaction vesselcentrifuged in a microfuge at 12,000 rpm for 5-10 seconds, decanted andresuspended into TE (500 U 1, 10 mM Tris-HCl, 1 mM EDTA pH 7.5) followedby recentrifugation. The cells were then diluted into TE (1 ml) andaliquots (200 μl) were spread onto the selective media previouslyprepared in 150 mm growth plates (VWR).

[0433] Alternatively, instead of multiple small reactions, thetransformation was performed using a single, large scale reaction,wherein reagent amounts were scaled up accordingly.

[0434] The selective media used was a synthetic complete dextrose agarlacking uracil (SCD-Ura) prepared as described in Kaiser et al., Methodsin Yeast Genetics, Cold Spring Harbor Press, Cold Spring Harbor, N.Y.,p. 208-210 (1994). Transformants were grown at 30° C. for 2-3 days.

[0435] The detection of colonies secreting amylase was performed byincluding red starch in the selective growth media. Starch was coupledto the red dye (Reactive Red-120, Sigma) as per the procedure describedby Biely et al., Anal. Biochem., 172:176-179 (1988). The coupled starchwas incorporated into the SCD-Ura agar plates at a final concentrationof 0.15% (w/v), and was buffered with potassium phosphate to a pH of 7.0(50-100 mM final concentration).

[0436] The positive colonies were picked and streaked across freshselective media (onto 150 mm plates) in order to obtain well isolatedand identifiable single colonies. Well isolated single colonies positivefor amylase secretion were detected by direct incorporation of redstarch into buffered SCD-Ura agar. Positive colonies were determined bytheir ability to break down starch resulting in a clear halo around thepositive colony visualized directly.

[0437] 4. Isolation of DNA by PCR Amplification

[0438] When a positive colony was isolated, a portion of it was pickedby a toothpick and diluted into sterile water (30 μl) in a 96 wellplate. At this time, the positive colonies were either frozen and storedfor subsequent analysis or immediately amplified. An aliquot of cells (5μl) was used as a template for the PCR reaction in a 25 μl volumecontaining: 0.5 μl Klentaq (Clontech, Palo Alto, Calif.); 4.0,u 110 mMdNTP's (Perkin Elmer-Celtus); 2.5 μl Kentaq buffer (Clontech); 0.25 μlforward oligo 1; 0.25 μl reverse oligo 2; 12.5 μl distilled water. Thesequence of the forward oligonucleotide 1 was:

[0439] 5′-TGTAAAACGACGGCCAGTTAAATAGACCTGCAATTATTAATCT-3′ (SEQ ID NO:169)

[0440] The sequence of reverse oligonucleotide 2 was:

[0441] 5′-CAGGAAACAGCTATGACCACCTGCACACCTGCAAATCCATT-3′ (SEQ ID NO: 170)

[0442] PCR was then performed as follows: a. Denature 92° C., 5 minutesb. 3 cycles of: Denature 92° C., 30 seconds Anneal 59° C., 30 secondsExtend 72° C., 60 seconds c 3 cycles of: Denature 92° C., 30 secondsAnneal 57° C., 30 seconds Extend 72° C., 60 seconds d. 25 cycles of:Denature 92° C., 30 seconds Anneal 55° C., 30 seconds Extend 72° C., 60seconds e. Hold 4° C.

[0443] The underlined regions of the oligonucleotides annealed to theADH promoter region and the amylase region, respectively, and amplifieda 307 bp region from vector pSST-AMY.0 when no insert was present.Typically, the first 18 nucleotides of the 5′ end of theseoligonucleotides contained annealing sites for the sequencing primers.Thus, the total product of the PCR reaction from an empty vector was 343bp. However, signal sequence-fused cDNA resulted in considerably longernucleotide sequences.

[0444] Following the PCR, an aliquot of the reaction (5 μl) was examinedby agarose gel electrophoresis in a 1% agarose gel using aTris-Borate-EDTA CFBE) buffering system as described by Sambrook et al.,supra. Clones resulting in a single strong PCR product larger than 400bp were further analyzed by DNA sequencing after purification with a 96Qiaquick PCR clean-up column (Qiagen Inc., Chatsworth, Calif.).

Example 3 Isolation of cDNA Clones Using Signal Algorithm Analysis

[0445] Various polypeptide-encoding nucleic acid sequences wereidentified by applying a proprietary signal sequence finding algorithmdeveloped by Genentech, Inc. (South San Francisco, Calif.) upon ESTs aswell as clustered and assembled EST fragments from public (e.g.,GenBank) and/or private (LIFESEQ®, Incyte Pharmaceuticals, Inc., PaloAlto, Calif.) databases. The signal sequence algorithm computes asecretion signal score based on the character of the DNA nucleotidessurrounding the first and optionally the second methionine codon(s)(ATG) at the 5′-end of the sequence or sequence fragment underconsideration. The nucleotides following the first ATG must code for atleast 35 unambiguous amino acids without any stop codons. If the firstATG has the required amino acids, the second is not examined. If neithermeets the requirement, the candidate sequence is not scored. In order todetermine whether the EST sequence contains an authentic signalsequence, the DNA and corresponding amino acid sequences; surroundingthe ATG codon are scored using a set of seven sensors (evaluationparameters) known to be associated with secretion signals. Use of thisalgorithm resulted in the identification of numerouspolypeptide-encoding nucleic acid sequences.

Example 4 Isolation of cDNA Clones Encoding Human PRO Polypeptides

[0446] Using the techniques described in Examples 1 to 3 above, numerousfull-length cDNA clones were identified as encoding PRO polypeptides asdisclosed herein. These cDNAs were then deposited under the terms of theBudapest Treaty with the American Type Culture Collection, 10801University Blvd., Manassas, Va. 20110-2209, USA (ATCC) as shown in Tabi7 below. TABLE 7 Material ATCC Dep. No. Deposit Date DNA26843-1389203099 Aug. 4, 1998 DNA30867-1335 209807 Apr. 28, 1998 DNA34431-1177209399 Oct. 17, 1997 DNA38268-1188 209421 Oct. 28, 1997 DNA40621-1440209922 Jun. 2, 1998 DNA40625-1189 209788 Apr. 21, 1998 DNA45409-2511203579 Jan. 12, 1999 DNA45495-1550 203156 Aug. 25, 1998 DNA49820-1427209932 Jun. 2, 1998 DNA56406-1704 203478 Nov. 17, 1998 DNA56410-1414209923 Jun. 2, 1998 DNA56436-1448 209902 May 27, 1998 DNA56855-1447203004 Jun. 23, 1998 DNA56860-1510 209952 Jun. 9, 1998 DNA56862-1343203174 Sep. 1, 1998 DNA56868-1478 203024 Jun. 23, 1998 DNA56869-1545203161 Aug. 25, 1998 DNA57704-1452 209953 Jun. 9, 1998 DNA58723-1588203133 Aug. 18, 1998 DNA57827-1493 203045 July 1, 1998 DNA58737-1473203136 Aug. 18, 1998 DNA58846-1409 209957 Jun. 9, 1998 DNA58850-1495209956 Jun. 9, 1998 DNA58855-1422 203018 Jun. 23, 1998 DNA59211-1450209960 Jun. 9, 1998 DNA59212-1627 203245 Sep. 9, 1998 DNA59213-1487209959 Jun. 9, 1998 DNA59605-1418 203005 Jun. 23, 1998 DNA59609-1470209963 Jun. 9, 1998 DNA59610-1556 209990 Jun. 16, 1998 DNA59837-2545203658 Feb. 9, 1999 DNA59844-2542 203650 Feb. 9, 1999 DNA59854-1459209974 Jun. 16, 1998 DNA60625-1507 209975 Jun. 16, 1998 DNA60629-1481209979 Jun. 16, 1998 DNA61755-1554 203112 Aug. 11, 1998 DNA62812-1594203248 Sep. 9, 1998 DNA62815-1576 203247 Sep. 9, 1998 DNA64881-1602203240 Sep. 9, 1998 DNA64886-1601 203241 Sep. 9, 1998 DNA64902-1667203317 Oct. 6, 1998 DNA64950-1590 203224 Sep. 15, 1998 DNA65403-1565203230 Sep. 15, 1998 DNA66308-1537 203159 Aug. 25, 1998 DNA66519-1535203236 Sep. 15, 1998 DNA66521-1583 203225 Sep. 15, 1998 DNA66658-1584203229 Sep. 15, 1998 DNA66660-1585 203279 Sep. 22, 1998 DNA66663-1598203268 Sep. 22, 1998 DNA66674-1599 203281 Sep. 22, 1998 DNA6886Z-2546203652 Feb. 9, 1999 DNA68866-1644 203283 Sep. 22, 1998 DNA68871-1638203280 Sep. 22, 1998 DNA68880-1676 203319 Oct. 6, 1998 DNA68883-1691203535 Dec. 15, 1998 DNA68885-1678 203311 Oct. 6, 1998 DNA71277-1636203285 Sep. 22, 1998 DNA73727-1673 203459 Nov. 3, 1998 DNA73734-1680203363 Oct. 20, 1998 DNA73735-1681 203356 Oct. 20, 1998 DNA76393-1664203323 Oct. 6, 1998 DNA77301-1708 203407 Oct. 27, 1998 DNA77568-1626203134 Aug. 18, 1998 DNA77626-1705 203536 Dec. 15, 1998 DNA81754-2532203542 Dec. 15, 1998 DNA81757-2512 203543 Dec. 15, 1998 DNA82302-2529203534 Dec. 15, 1998 DNA82340-2530 203547 Dec. 22, 1998 DNA83500-2506203391 Oct. 29, 1998 DNA84920-2614 203966 Apr. 27, 1999 DNA85066-2534203588 Jan. 12, 1999 DNA86571-2551 203660 Feb. 9, 1999 DNA87991-2540203656 Feb. 9, 1999 DNA92238-2539 203602 Jan. 20, 1999 DNA96042-2682PTA-382 Jul. 20, 1999 DNA96787-2534 203589 Jan. 12, 1999 DNA125185-2890PTA-1031 Dec. 7, 1999 DNA147531-2821 PTA-1185 Jan. 11, 2000DNA115291-2681 PTA-202 Jun. 8, 1999 DNA164625-28890 PTA-1535 Mar. 21,2000 DNA131639-2874 PTA-1784 Apr. 25, 2000 DNA79230-2525 203549 Dec. 22,1998

[0447] These deposits were made under the provisions of the BudapestTreaty on the International Recognition of the Deposit of Microorganismsfor the Purpose of Patent Procedure and the Regulations thereunder(Budapest Treaty). This assures maintenance of a viable culture of thedeposit for 30 years from the date of deposit. The deposits will be madeavailable by ATCC under the terms of the Budapest Treaty, and subject toan agreement between Genentech, Inc. and ATCC, which assures permanentand unrestricted availability of the progeny of the culture of thedeposit to the public upon issuance of the pertinent U.S. patent or uponlaying open to the public of any U.S. or foreign patent application,whichever comes first, and assures availability of the progeny to onedetermined by the U.S. Commissioner of Patents and Trademarks to beentitled thereto according to 35 USC §122 and the Commissioner's rulespursuant thereto (including 37 CFR §1.14 with particular reference to886 OG 638).

[0448] The assignee of the present application has agreed that if aculture of the materials on deposit should die or be lost or destroyedwhen cultivated under suitable conditions, the materials will bepromptly replaced on notification with another of the same. Availabilityof the deposited material is not to be construed as a license topractice the invention in contravention of the rights granted under theauthority of any government in accordance with its patent laws.

Example 5 Use of PRO as a hybridization probe

[0449] The following method describes use of a nucleotide sequenceencoding PRO as a hybridization probe.

[0450] DNA comprising the coding sequence of full-length or mature PROas disclosed herein is employed as a probe to screen for homologous DNAs(such as those encoding naturally-occurring variants of PRO) in humantissue cDNA libraries or human tissue genomic libraries.

[0451] Hybridization and washing of filters containing either libraryDNAs is performed under the following high stringency conditions.Hybridization of radiolabeled PRO-derived probe to the filters isperformed in a solution of 50% formamide, 5× SSC, 0.1% SDS, 0.1% sodiumpyrophosphate, 50 mM sodium phosphate, pH 6.8, 2× Denhardt's solution,and 10% dextran sulfate at 42° C. for 20 hours. Washing of the filtersis performed in an aqueous solution of 0.1× SSC and 0.1% SDS at 42° C.

[0452] DNAs having a desired sequence identity with the DNA encodingfull-length native sequence PRO can then be identified using standardtechniques known in the art.

Example 6 Expression of PRO in E. coli

[0453] This example illustrates preparation of an unglycosylated form ofPRO by recombinant expression in E. coli.

[0454] The DNA sequence encoding PRO is initially amplified usingselected PCR primers. The primers should contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector. A variety of expression vectors may be employed. Anexample of a suitable vector is pBR322 (derived from E. coli; seeBolivar et al., Gene, 2:95 (1977)) which contains genes for ampicillinand tetracycline resistance. The vector is digested with restrictionenzyme and dephosphorylated. The PCR amplified sequences are thenligated into the vector. The vector will preferably include sequenceswhich encode for an antibiotic resistance gene, a trp, promoter, apolyhis leader (including the first six STII codons, polyhis sequence,and enterokinase cleavage site), the PRO coding region, lambdatranscriptional terminator, and an argu gene.

[0455] The ligation mixture is then used to transform a selected E. colistrain using the methods described in Sambrook et al., supra.Transformants are identified by their ability to grow on LB plates andantibiotic resistant colonies are then selected. Plasmid DNA can beisolated and confirmed by restriction analysis and DNA sequencing.

[0456] Selected clones can be grown overnight in liquid culture mediumsuch as LB broth supplemented with antibiotics. The overnight culturemay subsequently be used to inoculate a larger scale culture. The cellsare then grown to a desired optical density, during which the expressionpromoter is turned on.

[0457] After culturing the cells for several more hours, the cells canbe harvested by centrifugation. The cell pellet obtained by thecentrifugation can be solubilized using various agents known in the art,and the solubilized PRO protein can then be purified using a metalchelating column under conditions that allow tight binding of theprotein.

[0458] PRO may be expressed in E. coli in a poly-His tagged form, usingthe following procedure. The DNA encoding PRO is initially amplifiedusing selected PCR primers. The primers will contain restriction enzymesites which correspond to the restriction enzyme sites on the selectedexpression vector, and other useful sequences providing for efficientand reliable translation initiation, rapid purification on a metalchelation column, and proteolytic removal with enterokinase. ThePCR-amplified, poly-His tagged sequences are then ligated into anexpression vector, which is used to transform an E. coli host based onstrain 52 (W3110 fuhA(tonA) lon galE rpoHts (htpRts) clpP(laclq).Transformants are first grown in LB containing 50 mg/ml carbenicillin at30° C. with shaking until an O.D.600 of 3-5 is reached. Cultures arethen diluted 50-100 fold into CRAP media (prepared by mixing 3.57 g(NH₄)₂SO₄; 0.71 g sodium citrate-2H2O, 1.07 g KCl, 5.36 g Difco yeastextract, 5.36 g Sheffield hycase SF in 500 mL water, as well as 110 mMMPOS, pH 7.3, 0.55% (w/v) glucose and 7 mM MgSO₄) and grown forapproximately 20-30 hours at 30° C. with shaking. Samples are removed toverify expression by SDS-page analysis, and the bulk culture iscentrifuged to pellet the cells. Cell pellets are frozen untilpurification and refolding.

[0459]E. coli paste from 0.5 to 1 L fermentations (6-10 g pellets) isresuspended in 10 volumes (w/v) in 7 M guanidine, 20 mM Tris, pH 8buffer. Solid sodium sulfite and sodium tetrathionate is added to makefinal concentrations of 0.1 M and 0.02 M, respectively, and the solutionis stirred overnight at 4° C. This step results in a denatured proteinwith all cysteine residues blocked by sulfitolization. The solution iscentrifuged at 40,000 rpm in a Beckman Ultracentifuge for 30 min. Thesupernatant is diluted with 3-5 volumes of metal chelate column buffer(6 M guanidine, 20 mM Tris, pH 7.4) and filtered through 0.22 micronfilters to clarify. The clarified extract is loaded onto a 5 ml QiagenNi-NTA metal chelate column equilibrated in the metal chelate columnbuffer. The column is washed with additional buffer containing 50 mMimidazole (Calbiochem, Utrol grade), pH 7.4. The protein is eluted withbuffer containing 250 mM imidazole. Fractions containing the desiredprotein are pooled and stored at 4° C. Protein concentration isestimated by its absorbance at 280 nm using the calculated extinctioncoefficient based on its amino acid sequence.

[0460] The proteins are refolded by diluting the sample slowly intofreshly prepared refolding buffer consisting of: 20 mM Tris, pH 8.6, 0.3M NaCl, 2.5 M urea, 5 mM cysteine, 20 mM glycine and 1 mM EDTA.Refolding volumes are chosen so that the final protein concentration isbetween 50 to 100 micrograms/ml. The refolding solution is stirredgently at 4° C. for 12-36 hours. The refolding reaction is quenched bythe addition of TFA to a final concentration of 0.4% (pH ofapproximately 3). Before further purification of the protein, thesolution is filtered through a 0.22 micron filter and acetonitrile isadded to 2-10% final concentration. The refolded protein ischromatographed on a Poros R1/H reversed phase column using a mobilebuffer of 0.1% TFA with elution with a gradient of acetonitrile from 10to 80%. Aliquots of fractions with A280 absorbance are analyzed on SDSpolyacrylamide gels and fractions containing homogeneous refoldedprotein are pooled. Generally, the properly refolded species of mostproteins are eluted at the lowest concentrations of acetonitrile sincethose species are the most compact with their hydrophobic interiorsshielded from interaction with the reversed phase resin. Aggregatedspecies are usually eluted at higher aceltonitrile concentrations. Inaddition to resolving misfolded forms of proteins from the desired form,the reversed phase step also removes endotoxin from the samples.

[0461] Fractions containing the desired folded PRO polypeptide arepooled and the acetonitrile removed using a gentle stream of nitrogendirected at the solution. Proteins are formulated into 20 mM Hepes, pH6.8 with 0.14 M sodium chloride and 4% mannitol by dialysis or by gelfiltration using G25 Superfine (Pharmacia) resins equilibrated in theformulation buffer and sterile filtered.

[0462] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 7 Expression of PRO in Mammalian Cells

[0463] This example illustrates preparation of a potentiallyglycosylated form of PRO by recombinant expression in mammalian cells.

[0464] The vector, pRK5 (see EP 307,247, published Mar. 15, 1989), isemployed as the expression vector. Optionally, the PRO DNA is ligatedinto pRK5 with selected restriction enzymes to allow insertion of thePRO DNA using ligation methods such as described in Sambrook et al.,supra. The resulting vector is called pRK5-PRO.

[0465] In one embodiment, the selected host cells may be 293 cells.Human 293 cells (ATCC CCL 1573) are grown to confluence in tissueculture plates in medium such as DMEM supplemented with fetal calf serumand optionally, nutrient components and/or antibiotics. About 10 μgpRK5-PRO DNA is mixed with about 1 μg DNA encoding the VA RNA gene[Thimmappaya et al., Cell, 31:543 (1982)] and dissolved in 500 μl of 1mM Tris-HCl, 0.1 mM EDTA, 0.227 M CaCl₂. To this mixture is added,dropwise, 500 U I of 50 mlM HEPES (pH 7.35), 280 mM NaCl, 1.5 mM NaPO 4,and a precipitate is allowed to form for 10 minutes at 25° C. Theprecipitate is suspended and added to the 293 cells and allowed tosettle for about four hours at 37° C. The culture medium is aspiratedoff and 2 ml of 20% glycerol in PBS is added for 30 seconds. The 293cells are then washed with serum free medium, fresh medium is added andthe cells are incubated for about 5 days.

[0466] Approximately 24 hours after the transfections, the culturemedium is removed and replaced with culture medium (alone) or culturemedium containing 200 μCi/ml ³⁵S-cysteine and 200 μCi/ml ³⁵S-methionine.After a 12 hour incubation, the conditioned medium is collected,concentrated on a spin filter, and loaded onto a 15% SDS gel. Theprocessed gel may be dried and exposed to film for a selected period oftime to reveal the presence of PRO polypeptide. The cultures containingtransfected cells may undergo further incubation (in serum free medium)and the medium is tested in selected bioassays.

[0467] In an alternative technique, PRO may be introduced into 293 cellstransiently using the dextran sulfate method described by Somparyrac etal., Proc. Natl. Acad. Sci., 12:7575 (1981). 293 cells are grown tomaximal density in a spinner flask and 700 μg pRK5-PRO DNA is added. Thecells are first concentrated from the spinner flask by centrifugationand washed with PBS. The DNA-dextran precipitate is incubated on thecell pellet for four hours. The cells are treated with 20% glycerol for90 seconds, washed with tissue culture medium, and re-introduced intothe spinner flask containing tissue culture medium, 5 μg/ml bovineinsulin and 0.1 μg/ml bovine transferrin. After about four days, theconditioned media is centrifuged and filtered to remove cells anddebris. The sample containing expressed PRO can then be concentrated andpurified by any selected method, such as dialysis and/or columnchromatography.

[0468] In another embodiment, PRO can be expressed in CHO cells. ThepRK5-PRO can be transfected into CHO cells using known reagents such asCaPO₄ or DEAE-dextran. As described above, the cell cultures can beincubated, and the medium replaced with culture medium (alone) or mediumcontaining a radiolabel such as ³⁵S-methionine. After determining thepresence of PRO polypeptide, the culture medium may be replaced withserum free medium. Preferably, the cultures are incubated for about 6days, and then the conditioned medium is harvested. The mediumcontaining the expressed PRO can then be concentrated and purified byany selected method.

[0469] Epitope-tagged PRO may also be expressed in host CHO cells. ThePRO may be subcloned out of the pRK5 vector. The subclone insert canundergo PCR to fuse in frame with a selected epitope tag such as apoly-his tag into a Baculovirus expression vector. The poly-his taggedPRO insert can then be subcloned into a SV40 driven vector containing aselection marker such as DHFR for selection of stable clones. Finally,the CHO cells can be transfected (as described above) with the SV40driven vector. Labeling may be performed, as described above, to verifyexpression. The culture medium containing the expressed poly-His taggedPRO can then be concentrated and purified by any selected method, suchas by Ni²⁺-chelate affinity chromatography.

[0470] PRO may also be expressed in CHO and/or COS cells by a transientexpression procedure or in CHO cells by another stable expressionprocedure.

[0471] Stable expression in CHO cells is performed using the followingprocedure. The proteins are expressed as an IgG construct(immunoadhesin), in which the coding sequences for the soluble forms(e.g. extracellular domains) of the respective proteins are fused to anIgG1 constant region sequence containing the hinge, CH2 and CH2 domainsand/or is a poly-His tagged form.

[0472] Following PCR amplification, the respective DNAs are subcloned ina CHO expression vector using standard techniques as described inAusubel et al., Current Protocols of Molecular Biology, Unit 3.16, JohnWiley and Sons (1997). CHO expression vectors are constructed to havecompatible restriction sites 5′ and 3′ of the DNA of interest to allowthe convenient shuttling of cDNA's. The vector used expression in CHOcells is as described in Lucas et al., Nucl. Acids Res. 24:9 (1774-1779(1996), and uses the SV40 early promoter/enhancer to drive expression ofthe cDNA of interest and dihydrofolate reductase (DHFR). DHFR expressionpermits selection for stable maintenance of the plasmid followingtransfection.

[0473] Twelve micrograms of the desired plasmid DNA is introduced intoapproximately 10 million CHO cells using commercially availabletransfection reagents Superfect® (Quiagen), Dosper® or Fugene®(Boehringer Mannheim). The cells are grown as described in Lucas et al.,supra. Approximately 3×10⁻⁷ cells are frozen in an ampule for furthergrowth and production as described below.

[0474] The ampules containing the plasmid DNA are thawed by placementinto water bath and mixed by vortexing. The contents are pipetted into acentrifuge tube containing 10 mLs of media and centrifuged at 1000 rpmfor 5 minutes. The supernatant is aspirated and the cells areresuspended in 10 mL of selective media (0.2 μm filtered PS20 with 5%0.2 μm diafiltered fetal bovine serum). The cells are then aliquotedinto a 100 mL spinner containing 90 mL of selective media. After 1-2days, the cells are transferred into a 250 mL spinner filled with 150 mLselective growth medium and incubated at 37° C. After another 2-3 days,250 mL, 500 mL and 2000 mL spinners are seeded with 3×10⁵ cells/mL. Thecell media is exchanged with fresh media by centrifugation andresuspension in production medium. Although any suitable CHO media maybe employed, a production medium described in U.S. Pat. No. 5,122,469,issued Jun. 16, 1992 may actually be used. A 3L production spinner isseeded at 1.2×10⁶ cells/mL. On day 0, the cell number pH is determined.On day 1, the spinner is sampled and sparging with filtered air iscommenced. On day 2, the spinner is sampled, the temperature shifted to33° C., and 30 mL of 500 g/L glucose and 0.6 mL of 10% antifoam (e.g.,35% polydimethylsiloxane emulsion, Dow Corning 365 Medical GradeEmulsion) taken. Throughout the production, the pH is adjusted asnecessary to keep it at around 7.2. After 10 days, or until theviability dropped below 70%, the cell culture is harvested bycentrifugation and filtering through a 0.22 μm filter. The filtrate waseither stored at 4° C. or immediately loaded onto columns forpurification.

[0475] For the poly-His tagged constructs, the proteins are purifiedusing a Ni-NTA column (Qiagen). Before purification, imidazole is addedto the conditioned media to a concentration of 5 mM. The conditionedmedia is pumped onto a 6 ml Ni-NTA column equilibrated in 20 mM Hepes,pH 7.4, buffer containing 0.3 M NaCl and 5 mM imidazole at a flow rateof 4-5 ml/min. at 4° C. After loading, the column is washed withadditional equilibration buffer and the protein eluted withequilibration buffer containing 0.25 M imidazole. The highly purifiedprotein is subsequently desalted into a storage buffer containing 10 mMHepes, 0.14 M NaCl and 4% mannitol, pH 6.8, with a 25 ml G25 Superfine(Pharmacia) column and stored at −80° C.

[0476] Immunoadhesin (Fc-containing) constructs are purified from theconditioned media as follows. The conditioned medium is pumped onto a 5ml Protein A column (Pharmacia) which had been equilibrated in 20 mM Naphosphate buffer, pH 6.8. After loading, the column is washedextensively with equilibration buffer before elution with 100 mM citricacid, pH 3.5. The eluted protein is immediately neutralized bycollecting 1 ml fractions into tubes containing 275 μl of 1 M Trisbuffer, pH 9. The highly purified protein is subsequently desalted intostorage buffer as described above for the poly-His tagged proteins. Thehomogeneity is assessed by SDS polyacrylamide gels and by N-terminalamino acid sequencing by Edman degradation.

[0477] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 8 Expression of PRO in Yeast

[0478] The following method describes recombinant expression of PRO inyeast.

[0479] First, yeast expression vectors are constructed for intracellularproduction or secretion of PRO from the ADH2/GAPDH promoter. DNAencoding PRO and the promoter is inserted into suitable restrictionenzyme sites in the selected plasmid to direct intracellular expressionof PRO. For secretion, DNA encoding PRO can be cloned into the selectedplasmid, together with DNA encoding the ADH2/GAPDH promoter, a nativePRO signal peptide or other mammalian signal peptide, or, for example, ayeast alpha-factor or invertase secretory signal/leader sequence, andlinker sequences (if needed) for expression of PRO.

[0480] Yeast cells, such as yeast strain AB110, can then be transformedwith the expression plasmids described above and cultured in selectedfermentation media. The transformed yeast supernatants can be analyzedby precipitation with 10% trichloroacetic acid and separation bySDS-PAGE, followed by staining of the gels with Coomassie Blue stain.

[0481] Recombinant PRO can subsequently be isolated and purified byremoving the yeast cells from the fermentation medium by centrifugationand then concentrating the medium using selected cartridge filters. Theconcentrate containing PRO may further be purified using selected columnchromatography resins.

[0482] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 9 Expression of PRO in Baculovirus-Infected Insect Cells

[0483] The following method describes recombinant expression of PRO inBaculovirus-infected insect cells.

[0484] The sequence coding for PRO is fused upstream of an epitope tagcontained within a baculovirus expression vector. Such epitope tagsinclude poly-his tags and immunoglobulin tags (like Fc regions of IgG).A variety of plasmids may be employed, including plasmids derived fromcommercially available plasmids such as pVL1393 (Novagen). Briefly, thesequence encoding PRO or the desired portion of the coding sequence ofPRO such as the sequence encoding the extracellular domain of atransmembrane protein or the sequence encoding the mature protein if theprotein is extracellular is amplified by PCR with primers complementaryto the 5′ and 3′ regions. The 5′ primer may incorporate flanking(selected) restriction enzyme sites. The product is then digested withthose selected restriction enzymes and subcloned into the expressionvector.

[0485] Recombinant baculovirus is generated by co-transfecting the aboveplasmid and BaculoGold™ virus DNA (Pharmingen) into Spodopterafrugiperda (“Sf9”) cells (ATCC CRL 1711) using lipofectin (commerciallyavailable from GIBCO-BRL). After 4-5 days of incubation at 28° C., thereleased viruses are harvested and used for further amplifications.Viral infection and protein expression are performed as described byO'Reilley et al., Baculovirus expression vectors: A Laboratory Manual,Oxford: Oxford University Press (1994).

[0486] Expressed poly-his tagged PRO can then be purified, for example,by Ni²⁺-chelate affinity chromatography as follows. Extracts areprepared from recombinant virus-infected Sf9 cells as described byRupert et al., Nature, 362:175-179 (1993). Briefly, Sf9 cells arewashed, resuspended in sonication buffer (25 mL Hepes, pH 7.9; 12.5 mMMgCl₂; 0.1 mM EDTA; 10% glycerol; 0.1% NP-40; 0.4 M KCl), and sonicatedtwice for 20 seconds on ice. The sonicates are cleared bycentrifugation, and the supernatant is diluted 50-fold in loading buffer(50 mM phosphate, 300 mM NaCl, 10% glycerol, pH 7.8) and filteredthrough a 0.45 μm filter. A Ni²⁺-NTA agarose column (commerciallyavailable from Qiagen) is prepared with a bed volume of 5 mL, washedwith 25 mL of water and equilibrated with 25 mL of loading buffer. Thefiltered cell extract is loaded onto the column at 0.5 mL per minute.The column is washed to baseline A₂₈₀ with loading buffer, at whichpoint fraction collection is started. Next, the column is washed with asecondary wash buffer (50 mM phosphate; 300 mM NaCl, 10% glycerol, pH6.0), which elutes nonspecifically bound protein. After reaching A₂₈₀baseline again, the column is developed with a 0 to 500 mM Imidazolegradient in the secondary wash buffer. One mL fractions are collectedand analyzed by SDS-PAGE and silver staining or Western blot withNi²⁺-NTA-conjugated to alkaline phosphatase (Qiagen). Fractionscontaining the eluted His₁₀-tagged PRO are pooled and dialyzed againstloading buffer.

[0487] Alternatively, purification of the IgG tagged (or Fc tagged) PROcan be performed using known chromatography techniques, including forinstance, Protein A or protein G column chromatography.

[0488] Many of the PRO polypeptides disclosed herein were successfullyexpressed as described above.

Example 10 Preparation of Antibodies that Bind PRO

[0489] This example illustrates preparation of monoclonal antibodieswhich can specifically bind PRO.

[0490] Techniques for producing the monoclonal antibodies are known inthe art and are described, for instance, in Goding, supra. Immunogensthat may be employed include purified PRO, fusion proteins containingPRO, and cells expressing recombinant PRO on the cell surface. Selectionof the immunogen can be made by the skilled artisan without undueexperimentation.

[0491] Mice, such as Balb/c, are immunized with the PRO immunogenemulsified in complete Freund's adjuvant and injected subcutaneously orintraperitoneally in an amount from 1-100 micrograms. Alternatively, theimmunogen is emulsified in MPL-TDM adjuvant (Ribi ImmunochemicalResearch, Hamilton, Mont.) and injected into the animal's hind footpads. The immunized mice are then boosted 10 to 12 days later withadditional immunogen emulsified in the selected adjuvant. Thereafter,for several weeks, the mice may also be boosted with additionalimmunization injections. Serum samples may be periodically obtained fromthe mice by retro-orbital bleeding for testing in ELISA assays to detectanti-PRO antibodies.

[0492] After a suitable antibody titer has been detected, the animals“positive” for antibodies can be injected with a final intravenousinjection of PRO. Three to four days later, the mice are sacrificed andthe spleen cells are harvested. The spleen cells are then fused (using35% polyethylene glycol) to a selected murine myeloma cell line such asP3X63AgU.1, available from ATCC, No. CRL 1597. The fusions generatehybridoma cells which can then be plated in 96 well tissue cultureplates containing HAT (hypoxanthine, aminopterin, and thymidine) mediumto inhibit proliferation of non-fused cells, myeloma hybrids, and spleencell hybrids.

[0493] The hybridoma cells will be screened in an ELISA for reactivityagainst PRO. Determination of “positive” hybridoma cells secreting thedesired monoclonal antibodies against PRO is within the skill in theart.

[0494] The positive hybridoma cells can be injected intraperitoneallyinto syngeneic Balb/c mice to produce ascites containing the anti-PROmonoclonal antibodies. Alternatively, the hybridoma cells can be grownin tissue culture flasks or roller bottles. Purification of themonoclonal antibodies produced in the ascites can be accomplished usingammonium sulfate precipitation, followed by gel exclusionchromatography. Alternatively, affinity chromatography based uponbinding of antibody to protein A or protein G can be employed.

Example 11 Purification of PRO Polypeptides Using Specific Antibodies

[0495] Native or recombinant PRO polypeptides may be purified by avariety of standard techniques in the art of protein purification. Forexample, pro-PRO polypeptide, mature PRO polypeptide, or pre-PROpolypeptide is purified by immunoaffinity chromatography usingantibodies specific for the PRO polypeptide of interest. In general, animmunoaffinity column is constructed by covalently coupling the anti-PROpolypeptide antibody to an activated chromatographic resin.

[0496] Polyclonal immunoglobulins are prepared from immune sera eitherby precipitation with ammonium sulfate or by purification on immobilizedProtein A (Pharmacia LKB Biotechnology, Piscataway, N.J.). Likewise,monoclonal antibodies are prepared from mouse ascites fluid by ammoniumsulfate precipitation or chromatography on immobilized Protein A.Partially purified immunoglobulin is covalently attached to achromatographic resin such as CnBr-activated SEPHAROSE™ (Pharmacia LKBBiotechnology). The antibody is coupled to the resin, the resin isblocked, and the derivative resin is washed according to themanufacturer's instructions.

[0497] Such an immunoaffinity column is utilized in the purification ofPRO polypeptide by preparing a fraction from cells containing PROpolypeptide in a soluble form. This preparation is derived bysolubilization of the whole cell or of a subcellular fraction obtainedvia differential centrifugation by the addition of detergent or by othermethods well known in the art. Alternatively, soluble PRO polypeptidecontaining a signal sequence may be secreted in useful quantity into themedium in which the cells are grown.

[0498] A soluble PRO polypeptide-containing preparation is passed overthe immunoaffinity column, and the column is washed under conditionsthat allow the preferential absorbance of PRO polypeptide (e.g., highionic strength buffers in the presence of detergent). Then, the columnis eluted under conditions that disrupt antibody/PRO polypeptide binding(e.g., a low pH buffer such as approximately pH 2-3, or a highconcentration of a chaotrope such as urea or thiocyanate ion), and PROpolypeptide is collected.

Example 12 Drug Screening

[0499] This invention is particularly useful for screening compounds byusing PRO polypeptides or binding fragment thereof in any of a varietyof drug screening techniques. The PRO polypeptide or fragment employedin such a test may either be free in solution, affixed to a solidsupport, borne on a cell surface, or located intracellularly. One methodof drug screening utilizes eukaryotic or prokaryotic host cells whichare stably transformed with recombinant nucleic acids expressing the PROpolypeptide or fragment. Drugs are screened against such transformedcells in competitive binding assays. Such cells, either in viable orfixed form, can be used for standard binding assays. One may measure,for example, the formation of complexes between PRO polypeptide or afragment and the agent being tested. Alternatively, one can examine thediminution in complex formation between the PRO polypeptide and itstarget cell or target receptors caused by the agent being tested.

[0500] Thus, the present invention provides methods of screening fordrugs or any other agents which can affect a PRO polypeptide-associateddisease or disorder. These methods comprise contacting such an agentwith a PRO polypeptide or fragment thereof and assaying (i) for thepresence of a complex between the agent and the PRO polypeptide orfragment, or (ii) for the presence of a complex between the PROpolypeptide or fragment and the cell, by methods well known in the art.In such competitive binding assays, the PRO polypeptide or fragment istypically labeled. After suitable incubation, free PRO polypeptide orfragment is separated from that present in bound form, and the amount offree or uncomplexed label is a measure of the ability of the particularagent to bind to PRO polypeptide or to interfere with the PROpolypeptide/cell complex.

[0501] Another technique for drug screening provides high throughputscreening for compounds having suitable binding affinity to apolypeptide and is described in detail in WO 84/03564, published on Sep.13, 1984. Briefly stated, large numbers of different small peptide testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. As applied to a PRO polypeptide, the peptide testcompounds are reacted with PRO polypeptide and washed. Bound PROpolypeptide is detected by methods well known in the art. Purified PROpolypeptide can also be coated directly onto plates for use in theaforementioned drug screening techniques. In addition, non-neutralizingantibodies can be used to capture the peptide and immobilize it on thesolid support.

[0502] This invention also contemplates the use of competitive drugscreening assays in which neutralizing antibodies capable of binding PROpolypeptide specifically compete with a test compound for binding to PROpolypeptide or fragments thereof. In this manner, the antibodies can beused to detect the presence of any peptide which shares one or moreantigenic determinants with PRO polypeptide.

Example 13 Rational Drug Design

[0503] The goal of rational drug design is to produce structural analogsof biologically active polypeptide of interest (i.e., a PRO polypeptide)or of small molecules with which they interact, e.g., agonists,antagonists, or inhibitors. Any of these examples can be used to fashiondrugs which are more active or stable forms of the PRO polypeptide orwhich enhance or interfere with the function of the PRO polypeptide invivo (c.f., Hodgson, Bio/Technology, 9: 19-21(1991)).

[0504] In one approach, the three-dimensional structure of the PROpolypeptide, or of a PRO polypeptide-inhibitor complex, is determined byX-ray crystallography, by computer modeling or, most typically, by acombination of the two approaches. Both the shape and charges of the PROpolypeptide must be ascertained to elucidate the structure and todetermine active site(s) of the molecule. Less often, useful informationregarding the structure of the PRO polypeptide may be gained by modelingbased on the structure of homologous proteins. In both cases, relevantstructural information is used to design analogous PRO polypeptide-likemolecules or to identify efficient inhibitors. Useful examples ofrational drug design may include molecules which have improved activityor stability as shown by Braxton and Wells, Biochemistry,31:7796-7801(1992) or which act as inhibitors, agonists, or antagonistsof native peptides as shown by Athauda et al., J. Biochem.,113:742-746(1993).

[0505] It is also possible to isolate a target-specific antibody,selected by functional assay, as described above, and then to solve itscrystal structure. This approach, in principle, yields a pharmacore uponwhich subsequent drug design can be based. It is possible to bypassprotein crystallography altogether by generating anti-idiotypicantibodies (anti-ids) to a functional, pharmacologically activeantibody. As a mirror image of a mirror image, the binding site of theanti-ids would be expected to be an analog of the original receptor. Theanti-id could then be used to identify and isolate peptides from banksof chemically or biologically produced peptides. The isolated peptideswould then act as the pharmacore.

[0506] By virtue of the present invention, sufficient amounts of the PROpolypeptide may be made available to perform such analytical studies asX-ray crystallography. In addition, knowledge of the PRO polypeptideamino acid sequence provided herein will provide guidance to thoseemploying computer modeling techniques in place of or in addition toX-ray crystallography.

Example 14 Pericyte c-Fos Induction (Assay 93)

[0507] This assay shows that certain polypeptides of the invention actto induce the expression of c-fos in pericyte cells and, therefore, areuseful not only as diagnostic markers for particular types ofpericyte-associated tumors but also for giving rise to antagonists whichwould be expected to be useful for the therapeutic treatment ofpericyte-associated tumors. Induction of c-fos expression in pericytesis also indicative of the induction of angiogenesis and, as such, PROpolypeptides capable of inducing the expression of c-fos would beexpected to be useful for the treatment of conditions where inducedangiogenesis would be beneficial including, for example, wound healing,and the like. Specifically, on day 1, pericytes are received from VECTechnologies and all but 5 ml of media is removed from flask. On day 2,the pericytes are trypsinized, washed, spun and then plated onto 96 wellplates. On day 7, the media is removed and the pericytes are treatedwith 100 μl of PRO polypeptide test samples and controls (positivecontrol=DME+5% serum+/−PDGF at 500 ng/ml; negative control=protein 32).Replicates are averaged and SD/CV are determined. Fold increase overProtein 32 (buffer control) value indicated by chemiluminescence units(RLU) luminometer reading verses frequency is plotted on a histogram.Two-fold above Protein 32 value is considered positive for the assay.ASY Matrix: Growth media=low glucose DMEM=20% FBS+1× pen strep+1×fungizone. Assay Media=low glucose DIMEM+5% FBS.

[0508] The following polypeptides tested positive in this assay: PRO1347and PRO1340.

Example 15 Ability of PRO Polypeptides to Stimulate the Release ofProteoglycans from Cartilage (Assay 97)

[0509] The ability of various PRO polypeptides to stimulate the releaseof proteoglycans from cartilage tissue was tested as follows.

[0510] The metacarphophalangeal joint of 4-6 month old pigs wasaseptically dissected, and articular cartilage was removed by free handslicing being careful to avoid the underlying bone. The cartilage wasminced and cultured in bulk for 24 hours in a humidified atmosphere of95% air, 5% CO₂ in serum free (SF) media (DME/F121:1) with 0.1% BSA and100U/ml penicillin and 100 μg/ml streptomycin. After washing threetimes, approximately 100 mg of articular cartilage was aliquoted intomicronics tubes and incubated for an additional 24 hours in the above SFmedia. PRO polypeptides were then added at 1% either alone or incombination with 18 ng/ml interleukin-1 α, a known stimulator ofproteoglycan release from cartilage tissue. The supernatant was thenharvested and assayed for the amount of proteoglycans using the1,9-dimethyl-methylene blue (DMB) colorimetric assay (Farndale andButtle, Biochem. BEiophys. Acta 883:173-177 (1985)). A positive resultin this assay indicates that the test polypeptide will find use, forexample, in the treatment of sports-related joint problems, articularcartilage defects, osteoarthritis or rheumatoid arthritis.

[0511] When various PRO polypeptides were tested in the above assay, thepolypeptides demonstrated a marked ability to stimulate release ofproteoglycans from cartilage tissue both basally and after stimulationwith interleukin-1α and at 24 and 72 hours after treatment, therebyindicating that these PRO polypeptides are useful for stimulatingproteoglycan release from cartilage tissue. As such, these PROpolypeptides are useful for the treatment of sports-related jointproblems, articular cartilage defects, osteoarthritis or rheumatoidarthritis. The polypeptides testing positive in this assay are: PRO1565,PRO1693, PRO1801 and PRO10096.

Example 16 Detection of Polypeptides That Affect Glucose or FFA Uptakein Skeletal Muscle (Assay 706)

[0512] This assay is designed to determine whether PRO polypeptides showthe ability to affect glucose or FFA uptake by skeletal muscle cells.PRO polypeptides testing positive in this assay would be expected to beuseful for the therapeutic treatment of disorders where either thestimulation or inhibition of glucose uptake by skeletal muscle would bebeneficial including, for example, diabetes or hyper- orhypo-insulinemia.

[0513] In a 96 well format, PRO polypeptides to be assayed are added toprimary rat differentiated skeletal muscle, and allowed to incubateovernight. Then fresh media with the PRO polypeptide and +/−insulin areadded to the wells. The sample media is then monitored to determineglucose and FFA uptake by the skeletal muscle cells. The insulin willstimulate glucose and FFA uptake by the skeletal muscle, and insulin inmedia without the PRO polypeptide is used as a positive control, and alimit for scoring. As the PRO polypeptide being tested may eitherstimulate or inhibit glucose and FFA uptake, results are scored aspositive in the assay if greater than 1.5 times or less than 0.5 timesthe insulin control.

[0514] The following PRO polypeptides tested positive as eitherstimulators or inhibitors of glucose and/or FFA uptake in this assay:PRO4405.

Example 17 Identification of PRO Polypeptides that Stimulate TNF-αRelease In Human Blood (Assay 728)

[0515] This assay shows that certain PRO polypeptides of the presentinvention act to stimulate the release of TNF-α in human blood. PROpolypeptides testing positive in this assay are useful for, among otherthings, research purposes where stimulation of the release of TNF-αwould be desired and for the therapeutic treatment of conditions whereinenhanced TNF-α release would be beneficial. Specifically, 200 μl ofhuman blood supplemented with 50 mM Hepes buffer (pH 7.2) is aliquottedper well in a 96 well test plate. To each well is then added 300 μlofeither the test PRO polypeptide in 50 mM Hepes buffer (at variousconcentrations) or 50 mM Hepes buffer alone (negative control) and theplates are incubated at 37° C. for 6 hours. The samples are thencentrifuged and 50 μl of plasma is collected from each well and testedfor the presence of TNF-α by ELISA assay. A positive in the assay is ahigher amount of TNF-α in the PRO polypeptide treated samples ascompared to the negative control samples.

[0516] The following PRO polypeptides tested positive in this assay:PRO263, PRO295, PRO1282, PRO1063, PRO1356, PRO3543, and PRO5990.

Example 18 Tumor Versus Normal Differential Tissue ExpressionDistribution

[0517] Oligonucleotide probes were constructed from some of the PROpolypeptide-encoding nucleotide sequences shown in the accompanyingfigures for use in quantitative PCR amplification reactions. Theoligonucleotide probes were chosen so as to give an approximately200-600 base pair amplified fragment from the 3′ end of its associatedtemplate in a standard PCR reaction. The oligonucleotide probes wereemployed in standard quantitative PCR amplification reactions with cDNAlibraries isolated from different human tumor and normal human tissuesamples and analyzed by agarose gel electrophoresis so as to obtain aquantitative determination of the level of expression of the PROpolypeptide-encoding nucleic acid in the various tumor and normaltissues tested. β-actin was used as a control to assure that equivalentamounts of nucleic acid was used in each reaction. Identification of thedifferential expression of the PRO polypeptide-encoding nucleic acid inone or more tumor tissues as compared to one or more normal tissues ofthe same tissue type renders the molecule useful diagnostically for thedetermination of the presence or absence of tumor in a subject suspectedof possessing a tumor as well as therapeutically as a target for thetreatment of a tumor in a subject possessing such a tumor. These assaysprovided the following results. Molecule is more highly expressed in: ascompared to: DNA26843-1389 normal lung lung tumor rectum tumor normalrectum DNA30867-1335 normal kidney kidney tumor DNA40621-1440 normallung lung tumor DNA40625-1189 normal lung lung tumor DNA45409-2511melanoma tumor normal skin DNA56406-1704 kidney tumor normal kidneynormal skin melanoma tumor DNA56410-1414 normal stomach stomach tumorDNA56436-1448 normal skin melanoma tumor DNA56855-1447 normal esophagusesophageal tumor rectum tumor normal rectum DNA56860-1510 normal kidneykidney tumor rectum tumor normal rectum DNA56862-1343 kidney tumornormal kidney normal lung lung tumor DNA56868-1478 normal stomachstomach tumor normal lung lung tumor DNA56869-1545 normal esophagusesophageal tumor normal skin melanoma tumor DNA57704-1452 normal stomachstomach tumor rectum tumor normal rectum DNA58723-1588 normal stomachstomach tumor kidney tumor normal kidney normal skin melanoma tumorDNA57827-1493 normal stomach stomach tumor normal skin melanoma tumorDNA58737-1473 esophageal tumor normal esophagus normal stomach stomachtumor DNA58846-1409 lung tumor normal lung DNA58850-1495 esophagealtumor normal esophagus kidney tumor normal kidney DNA58855-1422 normalstomach stomach tumor rectum tumor normal rectum DNA59211-1450 normalkidney kidney tumor DNA59212-1627 normal skin melanoma tumorDNA59213-1487 normal stomach stomach tumor normal skin melanoma tumorDNA59605-1418 melanoma tumor normal skin DNA59609-1470 esophageal tumornormal esophagus DNA59610-1556 esophageal tumor normal esophagus lungtumor normal lung normal skin melanoma tumor DNA59837-2545 normal skinmelanoma tumor DNA59844-2542 normal skin melanoma tumor esophageal tumornormal esophagus DNA59854-1459 normal esophagus esophageal tumor stomachtumor normal stomach normal lung lung tumor DNA60625-1507 normal lunglung tumor DNA60629-1481 normal esophagus esophageal tumor normal rectumrectum tumor DNA61755-1554 normal stomach stomach tumor kidney tumornormal kidney DNA62812-1594 normal stomach stomach tumor normal lunglung tumor normal rectum rectum tumor normal skin melanoma tumorDNA62815-1576 esophageal tumor normal esophagus DNA64881-1602 normalstomach stomach tumor normal lung lung tumor DNA64902-1667 esophagealtumor normal esophagus kidney tumor normal kidney DNA65403-1565 normalesophagus esophageal tumor DNA66308-1537 normal lung lung tumorDNA66519-1535 kidney tumor normal kidney DNA66521-1583 normal esophagusesophageal tumor normal stomach stomach tumor normal lung lung tumornormal rectum rectum tumor normal skin melanoma tumor DNA66658-1584normal lung lung tumor melanoma tumor normal skin DNA66660-1585 lungtumor normal lung DNA66674-1599 kidney tumor normal kidney normal lunglung tumor DNA68862-2546 melanoma tumor normal skin DNA68866-1644 normalstomach stomach tumor DNA68871-1638 lung tumor normal lung normal skinmelanoma tumor DNA68880-1676 normal lung lung tumor normal skin melanomatumor DNA68883-1691 esophageal tumor normal esophagus DNA68885-1678 lungtumor normal lung DNA71277-1636 normal stomach stomach tumorDNA73734-1680 normal lung lung tumor DNA73735-1681 esophageal tumornormal esophagus normal kidney kidney tumor lung tumor normal lungnormal skin melanoma tumor DNA76393-1664 esophageal tumor normalesophagus stomach tumor normal stomach lung tumor normal lung rectumtumor normal rectum DNA77568-1626 normal stomach stomach tumor lungtumor normal lung DNA77626-1705 normal rectum rectum tumor DNA81754-2532normal skin melanoma tumor DNA81757-2512 esophageal tumor normalesophagus normal stomach stomach tumor melanoma tumor normal skinDNA82302-2529 normal stomach stomach tumor normal lung lung tumorDNA82340-2530 normal esophagus esophageal tumor DNA85066-2534 lung tumornormal lung normal skin melanoma tumor DNA87991-2540 esophageal tumornormal esophagus DNA92238-2539 normal skin melanoma tumor DNA96787-2534normal kidney kidney tumor

Example 19 Identification of Receptor/Ligand Interactions

[0518] In this assay, various PRO polypeptides are tested for ability tobind to a panel of potential receptor or ligand molecules for thepurpose of identifying receptor/ligand interactions. The identificationof a ligand for a known receptor, a receptor for a known ligand or anovel receptor/ligand pair is useful for a variety of indicationsincluding, for example, targeting bioactive molecules (linked to theligand or receptor) to a cell known to express the receptor or ligand,use of the receptor or ligand as a reagent to detect the presence of theligand or receptor in a composition suspected of containing the same,wherein the composition may comprise cells suspected of expressing theligand or receptor, modulating the growth of or another biological orimmunological activity of a cell known to express or respond to thereceptor or ligand, modulating the immune response of cells or towardcells that express the receptor or ligand, allowing the preparation ofagonists, antagonists and/or antibodies directed against the receptor orligand which will modulate the growth of or a biological orimmunological activity of a cell expressing the receptor or ligand, andvarious other indications which will be readily apparent to theordinarily skilled artisan.

[0519] The assay is performed as follows. A PRO polypeptide of thepresent invention suspected of being a ligand for a receptor isexpressed as a fusion protein containing the Fc domain of human IgG (animmunoadhesin). Receptor-ligand binding is detected by allowinginteraction of the immunoadhesin polypeptide with cells (e.g. Cos cells)expressing candidate PRO polypeptide receptors and visualization ofbound immunoadhesin with fluorescent reagents directed toward the Fcfusion domain and examination by microscope. Cells expressing candidatereceptors are produced by transient transfection, in parallel, ofdefined subsets of a library of cDNA expression vectors encoding PROpolypeptides that may function as receptor molecules. Cells are thenincubated for 1 hour in the presence of the PRO polypeptideimmunoadhesin being tested for possible receptor binding. The cells arethen washed and fixed with paraformaldehyde. The cells are thenincubated with fluorescent conjugated antibody directed against the Fcportion of the PRO polypeptide immunoadhesin (e.g. FITC conjugated goatanti-human-Fc antibody). The cells are then washed again and examined bymicroscope. A positive interaction is judged by the presence offluorescent labeling of cells transfected with cDNA encoding aparticular PRO polypeptide receptor or pool of receptors and an absenceof similar fluorescent labeling of similarly prepared cells that havebeen transfected with other cDNA or pools of cDNA. If a defined pool ofcDNA expression vectors is judged to be positive for interaction with aPRO polypeptide immunoadhesin, the individual cDNA species that comprisethe pool are tested individually (the pool is “broken down”) todetermine the specific cDNA that encodes a receptor able to interactwith the PRO polypeptide immunoadhesin.

[0520] In another embodiment of this assay, an epitope-tagged potentialligand PRO polypeptide (e.g. 8 histidine “His” tag) is allowed tointeract with a panel of potential receptor PRO polypeptide moleculesthat have been expressed as fusions with the Fc domain of human IgG(immunoadhesins). Following a 1 hour co-incubation with the epitopetagged PRO polypeptide, the candidate receptors are eachimmunoprecipitated with protein A beads and the beads are washed.Potential ligand interaction is determined by western blot analysis ofthe immunoprecipitated complexes with antibody directed towards theepitope tag. An interaction is judged to occur if a band of theanticipated molecular weight of the epitope tagged protein is observedin the western blot analysis with a candidate receptor, but is notobserved to occur with the other members of the panel of potentialreceptors.

[0521] Using these assays, the following receptor/ligand interactionshave been herein identified:

[0522] (1) PRC)10272 binds to PRO5801.

[0523] (2) PRC)20110 binds to the human IL-17 receptor Yao et al.,Cytokine 9(11):794-800 (1997); also herein designated as PRO1) and toPRO20040.

[0524] (3) PRO10096 binds to PRO20233.

[0525] (4) PRO19670 binds to PRO1890.

[0526] The foregoing written specification is considered to besufficient to enable one skilled in the art to practice the invention.The present invention is not to be limited in scope by the constructdeposited, since the deposited embodiment is intended as a singleillustration of certain aspects of the invention and any constructs thatare functionally equivalent are within the scope of this invention. Thedeposit of material herein does not constitute an admission that thewritten description herein contained is inadequate to enable thepractice of any aspect of the invention, including the best modethereof, nor is it to be construed as limiting the scope of the claimsto the specific illustrations that it represents. Indeed, variousmodifications of the invention in addition to those shown and describedherein will become apparent to those skilled in the art from theforegoing description and fall within the scope of the appended claims.

1. An antibody that binds to the polypeptide shown in FIG. 12 (SEQ IDNO:12).
 2. The antibody of claim 1 which is a monoclonal antibody. 3.The antibody of claim 1 which is a humanized antibody.
 4. The antibodyof claim 1 which is an antibody fragment.
 5. The antibody of claim 1which is labeled.
 6. The antibody of claim 1 which specifically binds tothe polypeptide shown in FIG. 12 (SEQ ID NO:12).